Toxin Titers Research Articles

Effect of Preservatives on Shiga Toxigenic Phages and Shiga Toxin of Escherichia coli O157:H7

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring β-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.

Relationship between stx Genotype and Stx2 Expression Level in Shiga Toxin-Producing Escherichia coli O157 Strains

To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.

Live Attenuated Shigella dysenteriae Type 1 Vaccine Strains Overexpressing Shiga Toxin B Subunit

Shigella dysenteriae serotype 1 (S. dysenteriae 1) is unique among the Shigella species and serotypes in the expression of Shiga toxin which contributes to more severe disease sequelae and the ability to cause explosive outbreaks and pandemics. S. dysenteriae 1 shares characteristics with other Shigella species, including the capability of causing clinical illness with a very low inoculum (10 to 100 CFU) and resistance to multiple antibiotics, underscoring the need for efficacious vaccines and therapeutics. Following the demonstration of the successful attenuating capacity of deletion mutations in the guaBA operon in S. flexneri 2a vaccine strains in clinical studies, we developed a series of S. dysenteriae 1 vaccine candidates containing the fundamental attenuating mutation in guaBA. All strains are devoid of Shiga toxin activity by specific deletion of the gene encoding the StxA subunit, which encodes enzymatic activity. The StxB subunit was overexpressed in several derivatives by either plasmid-based constructs or chromosomal manipulation to include a strong promoter. All strains are attenuated for growth in vitro in the HeLa cell assay and for plaque formation and were safe in the Serény test and immunogenic in the guinea pigs. Each strain induced robust serum and mucosal anti-S. dysenteriae 1 lipopolysaccharide (LPS) responses and protected against wild-type challenge. Two strains engineered to overexpress StxB induced high titers of Shiga toxin neutralizing antibodies. These candidates demonstrate the potential for a live attenuated vaccine to protect against disease caused by S. dysenteriae 1 and potentially to protect against the toxic effects of other Shiga toxin 1-expressing pathogens.

Humoral immunity and injection-site reactions in cattle vaccinated with a multivalent clostridial vaccine administered via subcutaneous injection or via transdermal needle-free injection

To evaluate injection-site reactions and serum antibody titers in cattle vaccinated with a clostridial vaccine administered SC or via needle-free transdermal injection. Sixteen 11-to 12-month-old Herefords. Cattle in 2 groups were vaccinated on days 0 and 28 with a commercially available multivalent clostridial vaccine administered SC or transdermally Injection sites and serum antibody titers were evaluated at several time points after vaccination. Serum antibody titers against Clostridium perfringens beta toxin, Clostridium novyi alpha toxin, and Clostridium septicum alpha toxin were determined with an ELISA; Clostridium sordellii lethal toxin titers were determined with a toxin neutralization assay. Firm injection site swellings developed in cattle vaccinated via either route; however, at several observation times, swellings were significantly smaller in cattle vaccinated transdermally. Serum titers against C perfringens beta toxin and C septicum alpha toxin did not differ significantly between groups after vaccination; serum titers against C novyi alpha toxin were not significantly different between groups, except on days 10 and 56, when they were significantly higher in cattle vaccinated SC. Titers against C sordellii lethal toxin were significantly higher in cattle vaccinated SC on several days after vaccination, but titers were not significantly different after day 49. Transdermal vaccination of cattle resulted in serum antibody titers that were similar to those induced via SC vaccination and caused injection-site reactions that were significantly smaller. Transdermal vaccination may be an effective technique for vaccinating cattle against clostridial diseases while minimizing local reactions that often develop after clostridial vaccination.

Shiga Toxin-Producing Escherichia coli O100:H–: stx 2e in Drinking Water Contaminated by Waste Water in Finland

In November 2007, 450 m(3) of treated wastewater leaked into the drinking water distribution system contaminating the drinking water of over 10,000 inhabitants of Nokia, Southern Finland. Nearly 1,000 people visited the health centre because of gastroenteritis during the following 5 weeks. A wide range of enteric pathogens was found in the patients. The authors used the 16-plex PCR to investigate whether the five major diarrheagenic Escherichia coli pathotypes (EPEC, ETEC, STEC, EIEC or EAEC) were present in the contaminated drinking water and in the patients' stool samples. The contaminated drinking water was positive for genes characteristic of various E. coli pathotypes: pic, invE, hlyA, ent, escV, eae, aggR, stx(2) , estIa and astA. These genes, except stx(2), hlyA and invE, were also detected in the stool samples of the patients linked to this outbreak. A sorbitol positive, streptomycin resistant STEC strain was isolated from the drinking water, and belonged to the serotype O100:H(-), produced Stx2 toxin (titre 1:8 by reversed-passive latex agglutination method), and carried the genes stx(2e), estIa and irp2.

Role of rifampicin in limiting Escherichia coli O157:H7 Shiga-like toxin expression and enhancement of survival of infected BALB/c mice

The sequelae of infection with Escherichia coli O157:H7 include the potentially fatal haemolytic uraemic syndrome. The pathobiological process of E. coli O157:H7 is chiefly dependent on the production of Shiga-like toxins I and II (SLT-I and -II). Antibiotic treatment is currently refrained from since it may lead to enhanced release of SLTs from the bacterium. In this study, the potential utility of rifampicin in treating E. coli O157:H7 infections was assessed both in vitro and in vivo. Five strains of E. coli O157:H7 were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the transcription of the SLT-I- and SLT-II-encoding genes (stx1 and stx2, respectively). Treatment of bacterial strains with the rifampicin minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), or the MIC followed by the MBC led to the inhibition of stx1 and stx2 gene transcription. Treatment with the MIC or with the MIC followed by the MBC was also capable of limiting toxin release. SLT-I and SLT-II detection by reverse passive latex agglutination showed an effective decrease in toxin titres following treatment with the MIC of rifampicin or with the MIC followed by the MBC. Treatment of cultures with the MBC alone was not as effective in decreasing toxin titres. The efficacy of rifampicin in treating E. coli O157:H7-infected BALB/c mice was also assessed. Rifampicin treatment resulted in enhanced mouse survival and limited the weight loss of infected animals. In conclusion, both in vitro and in vivo tests showed that rifampicin may be useful in treating E. coli O157:H7 infection.

In vitro evaluation of Clostridium septicum alpha toxoid

Aiming to investigate in vitro alternatives, a test for neutralizing antibody detection using cell culture was developed. This test was more sensitive than previous animal models, allowing for detection of substantially lower alpha toxin and anti-alpha toxin titers. Titers observed during in vivo and in vitro seroneutralization had a correlation of 99.12%, indicating that cell culture is a viable alternative in the evaluation of vaccine potency, screening of vaccinal seeds, and Clostridium septicum alpha toxin titration.

Incidence and Reproduction Numbers of Pertussis: Estimates from Serological and Social Contact Data in Five European Countries

Despite large-scale vaccination programmes, pertussis has remained endemic in all European countries and has been on the rise in many countries in the last decade. One of the reasons that have been discussed for the failure of vaccination to eliminate the disease is continued circulation of the pathogen Bordetella pertussis by mostly asymptomatic and mild infections in adolescents and adults. To understand the impact of asymptomatic and undiagnosed infection on the transmission dynamics of pertussis we analysed serological data from five European countries in combination with information about social contact patterns from five of those countries to estimate incidence and reproduction numbers. We compared two different methods for estimating incidence from individual data on IgG pertussis toxin (PT) titres. One method combines the cross-sectional surveys of titres with longitudinal information about the distribution of amplitude and decay rate of titres in a back-calculation approach. The second method uses age-dependent contact matrices and cross-sectional surveys of IgG PT titres to estimate a next generation matrix for pertussis transmission among age groups. The next generation approach allows for computation of basic reproduction numbers for five European countries. Our main findings are that the seroincidence of infections as estimated with the first method in all countries lies between 1% and 6% per annum with a peak in the adolescent age groups and a second lower peak in young adults. The incidence of infections as estimated by the second method lies slightly lower with ranges between 1% and 4% per annum. There is a remarkably good agreement of the results obtained with the two methods. The basic reproduction numbers are similar across countries at around 5.5. Vaccination with currently used vaccines cannot prevent continued circulation and reinfection with pertussis, but has shifted the bulk of infections to adolescents and adults. If a vaccine conferring lifelong protection against clinical and subclinical infection were available pertussis could be eliminated. Currently, continuing circulation of the pathogen at a subclinical level provides a refuge for the pathogen in which it can evolve and adjust to infect vaccinated populations. Please see later in the article for the Editors' Summary.

Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.

Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests

In the hope of developing a vaccine against Clostridium difficile based on its toxin(s), we have developed a fermentation medium for the bacterium that results in the formation of Toxin A and contains no meat or dairy products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Soy Peptone A3, have been found to replace both the meat/dairy product tryptone in the preparation of working cell banks and seed media, and NZ-Soy BL4 does the same in the fermentation medium. These replacements yield even higher toxin titers.

Limited Survival of a Cry2Ab-Resistant Strain of <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae) on Bollgard II

Bollgard II cotton (which expresses two Bt insecticidal genes cry1Ac/cry2Ab) and conventional cotton, grown in the laboratory or field and sampled at different stages, was exposed to Helicoverpa armigera (Hübner) larvae of three genotypes: homozygous for resistance to Cry2Ab; homozygous for susceptibility to Cry2Ab, and heterozygous for resistance. Survival of all genotypes was limited on Bollgard II but increased as plants aged. This was particularly the case for homozygous resistant individuals, with 8.5% of this genotype surviving to pupation on mature cotton. The increasing survival is assumed to be caused by the decline in the titer of Cry1Ac toxin after flowering in Bollgard II because Cry2Ab homozygous resistant larvae can tolerate high levels of Cry2Ab toxin. Larvae heterozygous for resistance performed no better on Bollgard II than homozygous susceptible larvae. Survivors on Bollgard II grew more slowly and produced smaller pupae that yielded adults with reduced longevity and fecundity. When reared on conventional cotton, all genotypes generally performed equally, indicating an absence of fitness costs associated with Cry2Ab resistance under the conditions examined.

Activity of vancomycin against epidemic Clostridium difficile strains in a human gut model

Vancomycin and metronidazole remain the only primary options for the treatment of Clostridium difficile infection (CDI). Recent reports have suggested a superior clinical response to vancomycin therapy compared with metronidazole, but this has been difficult to prove or explain. There are few robust in vitro data of the effects of antibiotic treatment of CDI in a gut reflective setting. We used clindamycin to induce high-level toxin production by two epidemic C. difficile PCR ribotypes in a human gut model of CDI. Vancomycin was instilled into the models to achieve in vivo faecal concentrations. C. difficile populations and toxin titres, and gut bacterial populations and vancomycin levels were monitored before, during and after vancomycin instillation. Clindamycin treatment elicited C. difficile germination and high-level cytotoxin production. Vancomycin reduced total viable counts and cytotoxin titres of both C. difficile PCR ribotypes, with no evidence of recurrence before the model runs were ended. C. difficile PCR ribotype 027 populations exhibited greater germination capacity than did PCR ribotype 106. Vancomycin was more rapidly effective against the greater numbers of PCR ribotype 027 vegetative forms. Vancomycin showed no activity against C. difficile spores. Bacteriological response to vancomycin varies between strains causing CDI, possibly correlating with the extent of germination capacity. Vancomycin effectively reduced vegetative forms and cytotoxin titres of both of the epidemic C. difficile PCR ribotypes evaluated, but showed no anti-spore activity. Comparison with the results of a previous gut model study showed that vancomycin was more effective than metronidazole in reducing C. difficile PCR ribotype 027 numbers and cytotoxin titres.

Effect of metronidazole on growth and toxin production by epidemic Clostridium difficile PCR ribotypes 001 and 027 in a human gut model

We compared the behaviour of Clostridium difficile PCR ribotypes 001 and 027 in a human gut model, and compared the responses to metronidazole exposure. Using a human gut model primed with pooled human faeces, gut flora bacterial counts, C. difficile total viable counts, spore counts and cytotoxin titres were determined, following exposure to clindamycin, in the absence or presence of metronidazole. Duration of cytotoxin production by C. difficile ribotype 027 was markedly longer than that of ribotype 001 (23 versus 13 days, respectively), but peak toxin titres were similar. During toxin production, total C. difficile ribotype 027 populations had higher proportions of vegetative cells than did ribotype 001 (median 56.33 versus 23.54%). Similarly, total C. difficile ribotype 027 populations remained predominantly as vegetative cells for longer than did ribotype 001 (20 versus 9 days). The effects of metronidazole on C. difficile were markedly less than expected. Titres of C. difficile ribotype 001 cytotoxin were reduced but recurred following metronidazole administration. C. difficile ribotype 027 cytotoxin titres in the distal section of the gut model were unaffected by metronidazole. These observations correlated with poor metronidazole concentrations. Duration of cytotoxin production by C. difficile ribotype 027 markedly exceeds that of ribotype 001. Sub-optimal gut concentrations of metronidazole, possibly due to inactivation by components of normal gut flora, are associated with continued toxin production. These findings may help to explain the increased severity of symptoms and higher case-fatality ratio associated with infections due to C. difficile ribotype 027.

Clinical outcomes of intravenous immune globulin in severe clostridium difficile-associated diarrhea

Our objective was to determine if use of intravenous immune globulin (IVIG) decreases the incidence of mortality, colectomies, and length of stay in the hospital in patients presenting with severe Clostridium difficile-associated diarrhea (CDAD). A retrospective analysis was undertaken of 79 patients who had a positive C. difficile toxin titer and severe disease admitted to the University of Pittsburgh Medical Center Presbyterian between July 2001 and July 2003. Standard therapy for severe CDAD including intravenous metronidazole, oral vancomycin, or vancomycin enema was administered to all patients. Eighteen patients also received IVIG treatment (200-300 mg/kg); these were pair matched by propensity scoring with 18 patients who had the most similar characteristics and severity of CDAD from the available pool of 61 subjects who did not receive IVIG treatment. No significant difference was observed in the baseline characteristics between the two groups. There were no statistical differences in clinical outcomes as measured by all cause mortality, colectomies, and length of stay. These data demonstrate that the use of IVIG in severe CDAD remains unsubstantiated. This study, although limited by a small sample size, does not support the use of IVIG at this dose for severe CDAD outside of a controlled trial.

Blood Group, Immunity, and Risk of Infection with Vibrio cholerae in an Area of Endemicity

Individuals with blood group O are more susceptible than other individuals to severe cholera, although the mechanism underlying this association is unknown. To assess the respective roles of both intrinsic host factors and adaptive immune responses that might influence susceptibility to infection with Vibrio cholerae, we prospectively followed a cohort of household contacts of patients with cholera in Bangladesh. In this study, we made the novel observation that persons with blood group O were less likely than those with other blood groups to become infected with V. cholerae O1 (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.85; P = 0.008). Consistent with prior studies, however, household contacts with blood group O were more likely to develop severe illness if infected with V. cholerae O1 (OR, 2.3; 95% CI, 0.98 to 5.59; P = 0.05). While blood group O protected significantly against infection with V. cholerae O1, there was no evidence of protection against V. cholerae O139. A multivariate analysis demonstrated that the association between blood group O and protection from infection with V. cholerae O1 was independent of age, gender, and baseline anti-cholera toxin and vibriocidal antibody titers. Based on this epidemiologic evidence, we propose a hypothesis for understanding the association between blood group O and the risk of infection with V. cholerae O1 and O139 as well as the risk of developing severe symptoms once infected.

Comparison of the efficacy of ramoplanin and vancomycin in both in vitro and in vivo models of clindamycin-induced Clostridium difficile infection

Treatment of Clostridium difficile infection (CDI) is limited primarily to either metronidazole or vancomycin. We compared vancomycin and a novel glycolipodepsipeptide, ramoplanin, in both hamster and in vitro gut models of clindamycin-induced CDI. We used an in vitro triple-stage chemostat model that simulates the human gut, and an in vivo hamster model, both primed with clindamycin. Clindamycin exposure elicited symptomatic disease in the hamster model, and promoted C. difficile germination and toxin production in the gut model. C. difficile germination and toxin production were not associated with depletion of gut microflora in the gut model, but were temporarily associated with subinhibitory concentrations of clindamycin. Both ramoplanin and vancomycin were associated with rapid symptom resolution in the hamster model, and rapid toxin titre decrease in the in vitro gut model. In both models of CDI, vancomycin was associated with greater persistence of C. difficile spores. C. difficile spores were recovered significantly more often from the caecal contents of vancomycin-treated (n = 19/23) compared with ramoplanin-treated (n = 6/23) hamsters (P < 0.05). Results from the in vitro gut and hamster models were concordant. Ramoplanin and vancomycin were similarly effective at reducing cytotoxin production in the gut CDI model and in resolving symptoms in the hamster model. Ramoplanin may be more effective than vancomycin at killing spores and preventing spore recrudescence. These findings suggest a potential therapeutic role for ramoplanin in CDI that requires further clinical investigation.

Novel indica basmati line (B-370) expressing two unrelated genes of Bacillus thuringiensis is highly resistant to two lepidopteran insects in the field

We report the first field trials of indica basmati rice expressing two Bt genes, cry1Ac and cry2A simultaneously. Different transgenic lines were sown under field conditions for two consecutive years (2001 and 2002). Artificial infestation of yellow stem borer (YSB, Scirpophaga incertulas) and natural infestation of rice leaf folder (RLF, Cnaphalochrocus medinalis) were studied. Transgenic lines showed up to 100% and 98% resistance against YSB at vegetative and flowering stages, respectively, with 98% additional resistant against RLF as compared with the control. Variation in some morphological characteristics, e.g., the average number of tillers, plant height and maturity, were also observed. Transgenic lines produced up to 59% more grains than control plants under artificially augmented conditions, while up to an 8% increase was recorded under natural infestations. All lines expressed high level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits necessary to kill the target insects. It was also observed that the transgenic lines released Bt toxins from roots into Murashige and Skoog basal medium, hydroponic cultures and soil, which could be detected through sandwich ELISA. On the basis of these results these lines seem good candidates to be released as the first commercial cultivars of Bt indica basmati rice.

Effective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infusion) and dairy proteins or digests (e.g., casein hydrolysates)

We have developed a fermentation medium for Clostridium tetani that results in the formation of tetanus toxin and contains no meat (e.g., beef heart infusion) or dairy (e.g., casein digest) products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Quest Hy-Soy ®, have been found to replace both the meat extract and casein digest components of traditional tetanus toxin production media and to yield even higher toxin titers. The comparison of the traditional versus the new medium has been carried out repeatedly by us and the superiority of our medium has been consistently observed. To our knowledge, this is the first time that such a medium has been devised.

Release of Shiga Toxin by Membrane Vesicles in Shigella dysenteriae Serotype 1 Strains and In Vitro Effects of Antimicrobials on Toxin Production and Release

Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

Field evaluation and risk assessment of transgenic indica basmati rice

We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines.