Survey of Bacillus thuringiensis (Bt) Literature

Summary

We summarize the points made in the literature and remarks below:

• Bt is the same cellular organism as B. cereus (causes gastroenteritis) and B. anthracis (causes anthrax). The only difference is in one "organelle" of the cell, called a plasmid, that produces proteins. Each plasmid produces a unique protein that attacks a target life form. In the case of Bt, that life form is a caterpillar or other insect life forms.
• Can Bt become anthrax? Theoretically, yes - it could exchange a plasmid with a wild anthrax germ. Also, it is not easy to tell anthrax, B. cereus, and Bt apart - they look the same and grow in the same environments, and even test the same in some genetic tests. See this Seattle Weekly article for an interesting view.
• When Bt is sprayed, it can and does infect many people in the target zone, setting up colonies in their respiratory tract and maybe in their intestines that live for weeks or months. The effects of this are not known. Obvious symptoms do not appear, but what could happen years from now as a result of these infections is anyone's guess. It is suspected that "symptomless" infections can challenge the immune system and cause disease later (diabetes, multiple sclerosis, and lupus are some under consideration).
• There is growing evidence that exposure to inhaled bacterial antigens that generate immune responses in humans (a potential for allergic reactions) can trigger skin allergic reactons of various types in some individuals. Bt falls into this category. Epidemiological studies have so far almost completely ignored dermatological symptoms. There are a significant number of anecdotal reports of skin symptoms after sprayings of Bt products in New Zealand (see  New Zealand CC-PAM Health Incident Report at this link).
• The same Bt pesticides that are sprayed on crops produce detectable residues in foods, even processed foods such as pasta. Gastroenteritis (food poisoning or "stomach flu") can result. It is estimated that a sizeable percentage of such diseases that were previously thought to be caused by B. cereus are actually caused by Bt insecticides.
• People with sensitive immune systems could be affected in ways we do not yet know, but immune responses are seen when Bt infections establish in humans.
• Closing your house up will not prevent Bt from getting in. Staying indoors for even one day after a spraying will still expose you to significant amounts of Bt spores.
• Bt endotoxins may cause unexpected effects; for instance, some insects consuming genetically modified crops that produce Bt endotoxin actually grow faster. This could because the toxin is actually being used as food, but in experiments the insects had all the preferred food they could eat. An even more probable reason could be that Bt endotoxin acts as a hormone mimicing substance (this is not the first bacterial toxin to show this behavior). These types of substances (also called endocrine disrupters) are increasingly suspected of causing serious health problems in humans.

Can Bt live in Humans?

• The culture media used to grow Bt in the lab is the same media used to grow other human pathogenic bacteria.
• The conditions for growth of Bt (pH 7.4, temperature 37^oC, moist environment) are found in humans.
• Humans develop antibodies to the Bt organism.
• Even four months after a single exposure, Bt organisms of the same strain as the pesticide used in the exposure can be cultured from nasal swabs. It is unlikely that the original spores would still be present after this period of time.
• There are a few studies that show Bt canand does cause gastroenteritis in humans and that you can recover culturable Bt from nursery workers feces, indicating that Bt can live and grow in the intestinal tract.

Survey Papers

No Spray Zone has prepared survey paper, available here, that reviews the current literature (through 2005) on Bt and its human health effects. The paper assumes some knowledge of medical and biological terminology. In addition, a 2006 article published in the Journal of Pesticide Reform presents the information in the NSZ survey paper in a less technical form. You can find it here. Both of these articles have good bibliographies that are more exhaustive than the list below.

Sources
(In most cases, if a paper is cited, the abstract of the paper is given.)

Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides

Bernstein IL, Bernstein JA, Miller M, Tierzieva S, Bernstein DI, Lummus Z, Selgrade MK, Doerfler DL, Seligy VL Division of Immunology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. bernstil@email.uc.edu

Environ Health Perspect 1999 Jul;107(7):575-82

Although health risks to pesticides containing Bacillus thuringiensis (Bt) have been minimal, the potential allergenicity of these organisms has not been evaluated. Therefore, a health survey was conducted in farm workers before and after exposure to Bt pesticides. Farm workers who picked vegetables that required Bt pesticide spraying were evaluated before the initial spraying operation (n = 48) and 1 and 4 months after (n = 32 and 20, respectively). Two groups of low- (n = 44) and medium- (n = 34) exposure workers not directly exposed to Bt spraying were also assessed. The investigation included questionnaires, nasal/mouth lavages, ventilatory function assessment, and skin tests to indigenous aeroallergens and to a variety of Bt spore and vegetative preparations. To authenticate exposure to the organism present in the commercial preparation, isolates from lavage specimens were tested for Bt genes by DNA-DNA hybridization. Humoral immunoglobulin G (IgG) and immunoglobulin E (IgE) antibody responses to spore and vegetative Bt extracts were assayed. There was no evidence of occupationally related respiratory symptoms. Positive skin-prick tests to several spore extracts were seen chiefly in exposed workers. In particular, there was a significant (p < 0.05) increase in the number of positive skin tests to spore extracts 1 and 4 months after exposure to Bt spray. The number of positive skin test responses was also significantly higher in high (p < 0.05) than in low- or medium-exposure workers. The majority of nasal lavage cultures from exposed workers was positive for the commercial Bt organism, as demonstrated by specific molecular genetic probes. Specific IgE antibodies were present in more high-exposure workers (p < 0.05) than in the low and medium groups. Specific IgG antibodies occurred more in the high (p < 0.05) than in the low-exposure group. Specific IgG and IgE antibodies to vegetative organisms were present in all groups of workers. Exposure to Bt sprays may lead to allergic skin sensitization and induction of IgE and IgG antibodies, or both. 

Review of paper in AAAS Journal (Science), June 16, 2000, p. 288
From Appl. Environ. Microbiol. 66, 2627 (2000).

Remarkably, it appears that the ubiquitous soil-living bacterium and occasional food-poisoning culprit Bacillus cereus, the widely used insect biocontrol pathogen Bacillus thuringiensis, and the life-threatening biological warfare agent Bacillus anthracis are the same species, despite the striking differences in phenotype. The secret appears to lie in the plasmids harboured by B. anthracis and B. thuringiensis. When ten B. cereus-like strains were isolated for biochemical and genetic analysis from soil taken from anthrax outbreak sites, they were found by Helgason et al. to have the same chromosomal marker as the implicated B. anthracis strains, but no plasmids. In their natural environments these species have a relatively low rate of clone formation, and it is known that all three Bacillus species are naturally able to take up plasmids. Indeed, plasmid exchange between B. cereus and B. anthracis has been verified experimentally. However, before sounding a general alarm, it cannot be ruled out that there is some other special, but as yet undetected, feature of the B. anthracis genome that makes it alone of the three species particularly adept at retrieving and retaining virulence plasmids.

A complete physical map of a Bacillus thuringiensis chromosome

Carlson-CR; Kolsto-AB J-Bacteriol. 1993 Feb; 175(4): 1053-60

Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes. Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin. We conclude that B. thuringiensis is very closely related to B. cereus and that the distinction between B. cereus and B. thuringiensis should be reconsidered.

Bacillus thuringiensis serotype H34 isolated from human and insecticidal strains serotypes 3a3b and H14 can lead to death of immunocompetent mice after pulmonary infection

Hernandez,E; Ramisse,F; Cruel,T; le Vagueresse,R; Cavallo,JD

FEMS-Immunol-Med-Microbiol. 1999 May; 24(1): 43-7

In 1995, we isolated a strain of Bacillus thuringiensis serotype H34 from severe human tissue necrosis. This bacterium was able to induce myonecrosis in immunosuppressed mice after cutaneous infection. Its potential pathogenicity for immunocompetent hosts was investigated in a mouse model of pulmonary infection. Mice infected intranasally by a suspension containing 10(8) spores died within 8 h in a clinical toxic-shock syndrome. In the same conditions, infection with a mutant without crystalline toxin, with the supernatant from a culture containing 10⁸bacteria/ml and by the insecticidal strain serotypes 3a3b or H14 led to identical results. Lower inocula simply induced a local inflammatory reaction with bacterial persistence observed during the course of 10 days.

Public health implications of the microbial pesticide Bacillus thuringiensis: an epidemiological study, Oregon, 1985-86

Green-M; Heumann-M; Sokolow-R; Foster-LR; Bryant-R; Skeels-M Am-J-Public-Health. 1990 Jul; 80(7): 848-52

Bacillus thuringiensis var. kurstaki (B.t.-k) is a microbial pesticide which has been widely used for over 30 years. Its safety for a human population living in sprayed areas has never been tested. Surveillance for human infections caused by B.t.-k among Lane County, Oregon residents was conducted during two seasons of aerial B.t.-k spraying for gypsy moth control. Bacillus isolates from cultures obtained for routine clinical purposes were tested for presence of Bacillus thuringiensis (B.t.). Detailed clinical information was obtained for all B.t.-positive patients. About 80,000 people lived in the first year's spray area, and 40,000 in the second year's area. A total of 55 B.t.-positive cultures were identified. The cultures had been taken from 18 different body sites or fluids. Fifty-two (95 percent) of the B.t. isolates were assessed to be probable contaminants and not the cause of clinical illness. For three patients, B.t. could neither be ruled in nor out as a pathogen. Each of these three B.t.-positive patients had preexisting medical problems. The level of risk for B.t.-k and other existing or future microbial pesticides in immunocompromised hosts deserves further study.

Q-218 Comparative Cytotoxic Effects of Commercial Insecticidal Bacillus thuringiensis Products Using insect and Human Cell Bioassay.

A.F Tayabali, V.L. Seligy, H Health Canada, Ottowa, Ontario Canada.

Presented  at General Meeting, American Society of Microbiologists, May 1999, Chicago.

Quoting the study, "The patterns of damage to insect and human cells were the same". Their conclusion, "This study establishes for the first time the cytotoxic effects of commercial Bt products, which are largely target cell independent, resulting from an infection-like process."

Spatial and Temporal Distribution of Airborne Bacillus thuringiensis var. kurstaki during an Aerial Spray Program for Gypsy Moth Eradication

Kay Teschke,1,2 Yat Chow,2 Karen Bartlett,2 Andrew Ross,2 and Chris van Netten1,2
Address correspondence to K. Teschke, Department of Health Care and Epidemiology, Mather Building, 5804 Fairview Avenue, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
Telephone: (604) 822-2041. Fax: (604) 822-4994. E-mail: teschke@interchange.ubc.ca

From Environmental Health Perspectives Volume 109, Number 1, January 2001 Environ Health Perspect 109:47-54 (2001). [Online 12 December 2000] http://ehpnet1.niehs.nih.gov/docs/2001/109p47-54teschke/abstract.html

We measured airborne exposures to the biological insecticide Bacillus thuringiensis var. kurstaki (Btk) during an aerial spray program to eradicate gypsy moths on the west coast of Canada. We aimed to determine whether staying indoors during spraying reduced exposures, to determine the rate of temporal decay of airborne concentrations, and to determine whether drift occurred outside the spray zone. During spraying, the average culturable airborne Btk concentration measured outdoors within the spray zone was 739 colony-forming units (CFU)/m3 of air. Outdoor air concentrations decreased over time, quickly in an initial phase with a half time of 3.3 hr, and then more slowly over the following 9 days, with an overall half-time of about 2.4 days. Inside residences during spraying, average concentrations were initially 2-5 times lower than outdoors, but at 5-6 hr after spraying began, indoor concentrations exceeded those outdoors, with an average of 244 CFU/m3 vs. 77 CFU/m3 outdoors, suggesting that the initial benefits of remaining indoors during spraying may not persist as outside air moves indoors with normal daily activities. There was drift of culturable Btk throughout a 125- to 1,000-meter band outside the spray zone where measurements were made, a consequence of the fine aerosol sizes that remained airborne (count median diameters of 4.3 to 7.2 µm). Btk concentrations outside the spray zone were related to wind speed and direction, but not to distance from the spray zone.

"...several studies indicate that B.t.k. can be recovered from exposed mammals but that recovery decreases relatively fast after exposure is terminated. In this respect, the study by Oshodi and Macnaughtan (1990, Btk preparation manual by the manufacturer of Foray48B Novo Nordisk) is somewhat unusual. After inhalation exposure to Foray 48B, the numbers of viable B.t.k. spores recovered from the lungs [of mammals] did not decrease substantially during a 28-day observation period." (p. 4-6).

Isolation and characterization of Bacillus cereus-like bacteria from faecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides

International Archives of Occupational and Environmental Health, 2001

Gert B. Jensen1, , Preben Larsen2, Bodil L. Jacobsen3, Bodil Madsen3, Andrea Wilcks1, Lasse Smidt1 and Lars Andrup1

 (1) Department of Chemical Working Environment, National Institute of Occupational Health, Lerso Parkalle 105, 2100 Copenhagen, Denmark
 (2) Department of Occupational and Environmental Medicine, Odense University Hospital, Odense, Denmark
 (3) The Danish Veterinary and Food Administration, Soborg, Denmark

Abstract: Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B.  thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD). Methods: Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis.
Results: Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product.
Conclusions: The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate  found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces [emphasis NSZ].The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.

(10)

IDEAS AND PERSPECTIVES Could Bt transgenic crops have nutritionally favourable effects on resistant insects?

Ali H. Sayyed , Hugo Cerda  and Denis J. Wright

Ecology Letters, (2003) 6: 167-169 2003.Blackwell Publishing Ltd/CNRS

Abstract
We present an idea that larvae of some Bacillus thuringiensis (Bt ) resistant populations of the diamondback moth, Plutella xylostella (L.), may be able to use Cry1Ac toxin derived from Bt as a supplementary food protein. Bt transgenic crops could therefore have unanticipated nutritionally favourable effects, increasing the fitness of resistant populations. This idea is discussed in the context of the evolution of resistance to Bt transgenic crops.

(11)
This article specifically mentions Bt as a possible allergy trigger, citing the work in Bernstein, et al. above.

Young farmers with cellular reactivity to airborne microbes suffer more frequently from work-related skin symptoms and allergic dermatitis

R. Spiewak, C. Skorska, A. Góra1, A. Horoch, J. Dutkiewicz

Ann Agric Environ Med 2001, 8, 255–259.

Abstract: 75 farming students (49 males and 26 females aged 16–23 years) underwent dermatological, laryngological and pulmonary examination, skin prick tests with common and farm allergens, Phadiatop and total IgE measurement. After that, the migration inhibition tests with antigens of airborne microbes typical for farm environment (Saccharopolyspora rectivirgula, Pantoea agglomerans, and Aspergillus fumigatus) were carried out. Possible differences between students with positive results and those non-reactive were sought.
Results: 10 students reacted to at least one microbial antigen in the migration inhibition test. There were no significant differences in distribution of atopy, prick test results, total IgE, and Phadiatop between the reactive students and their classmates. Only one case of asthma was found, hence a further statistical analysis was not feasible. Allergic rhinitis has been found in 30% of the reactive and in 9.2% of non-reactive students; the difference, however, was not statistically significant (p = 0.06). Significant differences were found with respect to the frequency of allergic skin diseases (40% reactive versus 9.2% non-reactive, p = 0.009); no other triggering factors than microbial antigens could be identified in 2 out of 4 reactive students with dermatitis. Work-related symptoms were present in all reactive students (100% versus 27.7%, p = 0.001); 8 out of 10 reactive students did not show any other specific sensitisation. Antigens of airborne microbes are commonly associated with lung diseases. Our results, however, suggest that the skin may be affected as well. Relatively strong association between cellular reactivity to airborne microbes and skin diseases deserves further studies.