Human cell exposure assays of Bacillus thuringiensis commercial insecticides: production of Bacillus cereus-like cytolytic effects from outgrowth of spores

Most contemporary bioinsecticides are derived from scaled-up cultures of Bacillus thuringiensis subspecies israelensis (Bti) and kurstaki (Btk), whose particulate fractions contain mostly B. thuringiensis spores (> 10(12)/L) and proteinaceous aggregates, including crystal-like parasporal inclusion bodies (PIB). Based on concerns over relatedness to B. cereus-group pathogens, we conducted extensive testing of B. thuringiensis (BT) products and their subfractions using seven human cell types. The Bti/Btk products generated nonspecific cytotoxicities involving loss in bioreduction, cell rounding, blebbing and detachment, degradation of immunodetectable proteins, and cytolysis. Their threshold dose (Dt approximately equal.5 times 10(-14)% BT product/target cell) equated to a single spore and a target cell half-life (tLD(50)) of approximately 16 hr. At Dts > 10(4), the tLD(50) rapidly shifted to < 4 hr; with antibiotic present, no component, including PIB-related [delta]-endotoxins, was cytolytic up to an equivalent of approximately 10(9 )Dt. The cytolytic agent(s) within the Bti/Btk-vegetative cell exoprotein (VCP) pool is an early spore outgrowth product identical to that of B. cereus and acting possibly by arresting protein synthesis. No cytolytic effects were seen with VCP from B. subtilis and Escherichia coli. These data, including recent epidemiologic work indicate that spore-containing BT products have an inherent capacity to lyse human cells in free and interactive forms and may also act as immune sensitizers. To critically impact at the whole body level, the exposure outcome would have to be an uncontrolled infection arising from intake of Btk/Bti spores. For humans, such a condition would be rare, arising possibly in equally rare exposure scenarios involving large doses of spores and individuals with weak or impaired microbe-clearance capacities and/or immune response systems.     

Isolation and characterization of novel human parechovirus from clinical samples

Using Vero cells, we isolated a virus (NII561-2000) from a cerebrospinal fluid specimen of a 1-year-old girl with Reye syndrome. The determined amino acid sequence of the virus indicated that the isolate was a human parechovirus (HPeV), a member of Picornaviridae. Neutralization test showed that the NII561-2000 virus had distinct antigenicity to HPeV-1, HPeV-2, and HPeV-3, and that the sequence was distinct from these types as well as from HPeV-4 and HPeV-5. Thus, we propose the virus (NII561-2000) as the prototype of HPeV-6. We isolated 10 NII561-2000-related viruses, 14 HPeV-1, 16 HPeV-3, and 1 HPeV-4 of 41 HPeVs from various clinical samples collected in Niigata, Japan. Clinical symptoms of the persons infected with the NII561-2000-related viruses were infectious gastroenteritis, rash, upper respiratory tract infection, and paralysis, in addition to Reye syndrome in the 1-year-old girl.     

Biological monitoring of exposure to organophosphorus insecticides in a group of horticultural greenhouse workers

Exposure to selected organophosphorus insecticides (OPs), malathion, diazinon and acephate, was evaluated in a group of horticultural greenhouse workers. This was achieved through measurements of the cumulative urinary excretion time courses of specific and non-specific biomarkers over a 24 h period following the onset of work exposure. For malathion, the absorbed daily doses were estimated from the 24 h cumulative urinary amounts of the specific mono- and di-carboxylic acid metabolites (the sum of MCA and DCA) through the use of a kinetic model. The observed 24 h urinary levels were also compared with a biological reference value (BRV) of 57 nmol kg(-1) of body weight established in a previous work on the basis of a human no-observed-effect level exposure dose. Excretion values were found to be 2.5% or less of the BRV, suggesting a negligible health risk. Both median and 95th percentile concentrations of DCA (n = 57 samples) were, however, slightly higher than the baseline values determined by the Centers for Disease Control and Prevention (CDC) in the US civilian population (MCA was not analyzed by the CDC). The cumulative urinary excretion time course of the methyl phosphoric (MP) derivatives, which are metabolites of malathion but also of several other OPs, was also determined. Though relatively low, the MP levels were from 3 to 31 times higher than would be expected on the basis of the malathion specific MCA and DCA excretions, indicating that MP excretions stem from sources other than malathion exposure. Accordingly, only the time courses of MCA and DCA excretion rate (nmol h(-1)) were compatible with the time of work exposure. Urinary biomarkers of exposure to diazinon and acephate were also measured. Urinary concentrations were essentially below or equal to the analytical limit of detection of 1 microg l(-1) for 2-isopropyl-4-methyl-6-hydroxypyrimidine (n = 54) and of 0.8 microg l(-1) for acephate and methamidophos (n = 59): values within the baseline range of the US civilian population, like the observed phosphoric metabolite concentrations. The workers under study thus appeared to be only slightly more exposed to malathion than the general population. However, their overall exposure to OPs, as measured by non-specific phosphoric metabolites, was similar to that of the general population, whose exposure occurs mainly through the ingestion of contaminated food. These results question the relevance of measuring non-specific phosphoric metabolites when attempting to assess low-dose occupational exposure to a specific OP.     

Isolation and molecular characterization of Escherichia coli O157 from broiler and human samples

There is a lack of information about the role of poultry, specifically chicken, in transmission of Escherichia coli (E. coli) O157 and subsequent human illnesses. This study was therefore aimed at investigating the presence of E. coli O157 and its virulence genes in various samples collected from broiler chickens and humans in Eastern Turkey by culture, immunomagnetic separation (IMS), and polymerase chain reaction (PCR). The genetic relationship between broiler and human isolates was also examined by pulsed-field gel electrophoresis (PFGE). In the PCR analysis of sorbitol-negative isolates, E. coli O157 was identified in 0.1% (1/1000) and 0.4% (4/1000) of the liver and cecum samples of broiler chickens, respectively. On the other hand, none of the carcass samples were determined to be positive for E. coli O157. Overall, the results indicated that 12% (3/25) of the flocks were positive for E. coli O157. The differences between the flocks in terms of the positivity were determined to be statistically significant (p<0.001). Ten (2.7%) of 367 human stool samples were also positive for E. coli O157 in the PCR examination. None of the broiler and human E. coli O157 isolates possessed H7, shigatoxins 1-2, or enterohemolysin genes, whereas all the broiler isolates and one of the human isolates were positive for intimin gene. In the PFGE analysis, a total of eight different profiles (four from broiler and four from human isolates) were observed. However, there were no genetic relationships between broiler and human E. coli O157 isolates. It can be concluded that more detailed studies are needed in poultry to better understand the role of these species in the epidemiology of E. coli 0157 infections in humans.     

Identification and antibiotic resistance of faecal enterococci isolated from water samples

We have isolated 47 strains of presumptive faecal streptococci from different water samples. Identification was made by the method of Facklam et al. (1989). Antibiotic resistance was studied on Mueller-Hinton Agar. Twelve antibiotics were tested. High-level aminoglycoside resistance (HLAR) and resistance to glycopeptides were studied. Biochemical identification of presumptive faecal streptococci isolates gave the following results: 19 Enterococcus faecalis, 12 E. faecium, 8 E. hirae, 4 E. durans and 4 E. mundtii. E. mundtii is not included among faecal enterococci. None of the strains were resistant to glycopeptides (vancomycin and teicoplanin). Three strains of Enterococci showed HLAR. Two of them were isolated from coastal bathing waters and the other from wastewater. This suggests that water could contribute to spread of HLAR enterococci and it should be a matter of concern for public health authorities.     

Health effects and personal protection in pest control workers using organophosphorus insecticides]

One hundred and sixteen pest control workers (16 termite control workers (Sprayer I), 46 cockroach or fly-control workers (Sprayer II) and 56 both termite and cockroach or fly-control workers (Sprayer III)) were surveyed by a questionnaire and health examinations. Variety and quantity of pesticide used, personal usage of protective devices (respirator, gloves, work clothes, under wear), and conditions related to bathing were studied by a questionnaire. Relationships between protective devices and blood cholinesterase activity (ChE) were also analyzed. Significant findings were as follows: Most of the pest control operators wore gum gloves and gas masks when involved in termite control. However in the case of cockroach or fly-control, 38% did not wear gloves, and for respiratory protection 44% wore gas masks while 25% of them did not even use any form of respirators. In spite of having better protective practices than sprayers II and III, sprayer I showed significantly lower plasma ChE activity than both sprayer II and III. Plasma ChE activity in sprayers who put on simple respirators tended to be lower than those using gas masks. Plasma ChE activity was also significantly lower in those workers who had less frequent changes of work clothes compared to those with more frequent.     

Lead exposure in female workers who are pregnant or of childbearing age

In adults, high-level lead exposure often occurs in the lead-related industries. Acute lead poisoning has become rare, but chronic exposure to low-level lead remains a public health issue. With recent advances in our understanding of lead toxicity at low-levels, researchers have shifted their focus to studying lead at concentrations below those currently recommended as 'acceptable' in worker protection. As gender plays an important role in the storage, biokinetics, and toxicity of lead, it seems inappropriate to extrapolate findings of lead exposure in men to women. Women's bones release lead more slowly to the bloodstream, so blood levels remain increased for a long time after cessation of high exposure, reflecting the endogenous source of the lead. Particularly in pregnant women, bone lead release could influence health in pregnancy and be extremely harmful to the rapidly growing and developing fetus. Accordingly, female workers of childbearing age should avoid excessive lead exposure. However, because studies of pregnant workers encounter many difficulties and inconveniences, sufficient research has not been conducted in this area. As an alternative, a group of non-occupationally exposed women, matched as well as possible for anthropometric and reproductive variables and with almost the same levels of blood lead, could be recruited for survey.     

Isolation of Salmonella from meat samples and characterization by enterobacterial repetitive intergenic consensus-polymerase chain reaction and antibiotics test

Salmonella is one of the most important foodborne pathogens associated with severe diseases in animals and humans. Meat samples are considered as one of the main sources of Salmonella infections. Consequently, the survey of Salmonella contamination in meat samples is of outmost importance for the control and prevention of severe diseases. In this study, 250 meat samples were selected for surveys of Salmonella contaminations. Results indicated that 12% (n=30) of samples tested were positive to Salmonella. The genetic characterization of 30 Salmonella was studied by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and 22 of ERIC-PCR types were found with D of 94.8%. In addition, the resistant characterization was also carried out using nine antibiotics test, and nine resistant patterns were observed with D of 88.7%. A good correlation was also observed between ERIC-PCR fingerprinting and resistant patterns in some Salmonella such as SAL 6 and SAL 7.     

PCR detection of Chlamydia psittaci in faecal samples from passerine birds in Sweden.

To investigate to what extent wild passerine birds are carriers of Chlamydia psittaci, 312 faecal samples from 18 bird species were collected. Using the PCR technique and subsequent DNA sequencing, C. psittaci DNA was demonstrated in faecal samples from 9 (2.9%) birds of 6 different species. Sera from 65 bird-ringers, highly exposed to wild birds, were tested by microimmunofluorescence assay for the occurrence of IgG and IgM antibodies to C. psittaci. No such antibodies were found. This results indicate that a significant proportion of wild passerine birds are carriers of C. psittaci, but rarely infectious to humans.     

Escherichia coli populations from diabetic and non-diabetic patients with bacteraemia and faecal samples from healthy subjects--a comparative study

Biochemical reactions, using the PhP-EC system of biochemical fingerprinting, were evaluated in order to group strains into different clusters and to investigate whether a biochemical typing system may be used to distinguish between randomly selected Escherichia coli strains obtained from bacteraemic patients and healthy controls. Fifty epidemiologically unrelated strains isolated from blood of non-diabetic patients and 46 faecal control strains were studied. Separately, 70 E. coli strains from 64 diabetic patients with bacteraemia were investigated. Diversity index was 0.977, 0.969 and 0.941 respectively. The strains were clustered at a similarity level of 0.95. The bacteraemic and control strains were subdivided into 14 different clusters with 2-12 strains each and 40 'outliers'. The largest cluster was dominated by bacteraemic strains (9/12, 75%). Two other clusters were dominated by control strains. In the remaining groups blood and faecal isolates were evenly distributed. No biochemical test was able to distinguish between bacteraemic and faecal control strains. Strains from patients with diabetes mellitus were grouped in 11 clusters containing 2-14 strains and 22 'outliers'. The low diversity index of bacteraemic strains obtained from diabetic patients as compared to other strains indicated a greater homogenicity. However, no correlation was observed between the examined host factors and the clusters.     

Mortality from cancer and chronic respiratory diseases among workers who manufacture carbon electrodes

OBJECTIVES—To investigate the risk of cancer and non-neoplastic respiratory diseases among workers who manufacture carbon electrodes, as this industry entails exposure to mixtures of polycyclic aromatic hydrocarbons.
METHODS—A historical cohort study was carried out of 1006 male workers employed for at least 1 year between 1945 and 1971 in a carbon (graphite) electrode production plant in central Italy, who were followed up for mortality between 1955 and 1996. The ratio of observed to expected deaths (standardised mortality ratios, SMRs) was computed from both national and (for the period 1964-96) regional age and period specific mortalities. A multivariate Poisson regression analysis was performed to investigate the relative risk (RR) of death according to duration of employment and time since first employment in the factory.
RESULTS—A total of 424 workers had died, 538 were still alive, and 44 were lost to follow up. Mortalities from all causes, all cancers, and respiratory tract cancer were in line with the regional figure. An excess was found over the expected deaths from skin cancer including melanoma (SMR 3.16, 95% confidence interval (95% CI) 0.65 to 9.23) and from non-neoplastic respiratory diseases (SMR 1.58, 95% CI 1.16 to 2.11). Poisson regression analysis including age as a covariate showed an increased risk of dying from gastric cancer with increasing duration of employment, and an increase in the RR of dying from lung cancer and from non-neoplastic respiratory diseases with increasing time since first employment, although the linear trend was not significant.
CONCLUSION—This study supports previous findings that working in the carbon electrode manufacturing industry may not increase the risk of dying from respiratory cancer. However, a possible association with non-malignant respiratory diseases cannot be excluded.


Keywords: carbon electrode manufacturing; polycyclic aromatic hydrocarbons; historical cohort study     

Isolation of mosquito-toxic bacteria from mosquito-breeding sites in Kenya.

A large number of source materials were collected for isolating entomopathogenic bacteria from larval mosquito habitats in Kirinyaga District, Kenya. Mosquito-toxic bacteria were included among the numerous types of microorganisms isolated from the habitats. The pathogenic isolates shared common structural characteristics; they were gram-positive, spore-forming bacilli that produced parasporal inclusions conferring broad-spectrum larvicidal activity against Anopheles, Culex and Aedes mosquitoes. Based on structural and growth characteristics, coupled with larvicidal activity, the pathogenic isolates were tentatively identified as variants of Bacillus thuringiensis. Although the collection consisted of a variety of items including soil, silt and mud, the most productive materials were larval bodies. Using healthy mosquito larvae held in a fully permeable plastic bottle, a baiting technique was developed as a means of recovering bacteria from the environment.     

Exposure of field workers to organophosphorus insecticides: Sweet corn and peaches

Plasma and red blood cell cholinesterase levels of professional agricultural workers engaged in packing sweet corn and thinning peaches were monitored. Workers with extensive contact with mechanically harvested sweet corn (the corn had been treated one or two days before harvest with a combination of ethyl and methyl parathion) exhibited significant depression of cholinesterase. Gloves, worn by 40% of the workers, provided some protection from absorption of pesticide residues. No significant cholinesterase depression was found in workers thinning peaches which had been previously treated with parathion.