The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants' sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants < or = 2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4-6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.     

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/ 19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (chi2 = 5.42, P < 0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in > 50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.     

Distribution and selective elimination of paralytic shellfish toxins in different tissues of Octopus vulgaris

Octopus (Octopus vulgaris, Cuvier) plays a central role in the marine food web, being an important consumer with high metabolic rates and at the same time an important food item for higher predators. After harmful algal blooms, octopus can accumulate high levels of marine toxins trough trophic interrelationships. The aim of this study is to characterize the distribution of paralytic shellfish toxins (PSTs) in selected tissues of the O. vulgaris, in order to assess the translocation of toxins among organs with different physiological functions. Different retention times and selective elimination of particular toxin analogues were also investigated. Twenty three specimens of O. vulgaris were captured in Peniche (NW coast of Portugal) after PSTs have been detected in molluscan bivalves. Tissue matrices were dissected from organs with digestive function (digestive gland, stomach and salivary glands) and excretory function (kidneys and branchial hearts) and analyzed for toxin determination. Toxin analysis was carried out by high performance liquid chromatography with fluorescence detection (LC-FLD). PSTs were found in all tissues analyzed. The highest toxin concentrations were found in the digestive gland, reaching a maximum of 2980 μg STX equiv. kg⁻¹. The toxin profile was constituted by dcSTX, B1, C1 + 2, dcGTX2 + 3, dcNEO, STX and GTX2 + 3. A lower number of toxins were identified in the remaining organs, with B1 and dcSTX compromising more than 90% in molar fraction. Decarbamoyl saxitoxin was the most abundant toxin detected in digestive gland, stomach and salivary glands, while B1 was dominant in organs with excretory function. A positive correlation of concentrations of B1 and dcSTX were found in the organs analyzed. Results indicate that B1 and dcSTX are assimilated into the digestive gland in a similar proportion. Selective elimination of toxins with higher elimination of B1 and retention of dcSTX is suggested. This study contributes to better understanding of the dynamics of PSTs in O. vulgaris and the fate of PSTs in the food web.     

PHOTOSYNTHETIC PIGMENT AND GENETIC DIFFERENCES BETWEEN TWO SOUTHERN OCEAN MORPHOTYPES OF EMILIANIA HUXLEYI (HAPTOPHYTA)1

The widespread coccolithophorid Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler plays a pivotal role in the carbon pump and is known to exhibit significant morphological, genetic, and physiological diversity. In this study, we compared photosynthetic pigments and morphology of triplicate strains of Southern Ocean types A and B/C. The two morphotypes differed in width of coccolith distal shield elements (0.11–0.24 μm, type A; 0.06–0.12 μm, type B/C) and morphology of distal shield central area (grill of curved rods in type A; thin plain plate in type B/C) and showed differences in carotenoid composition. The mean 19′‐hexanoyloxyfucoxanthin (Hex):chl a ratio in type B/C was >1, whereas the type A ratio was <1. The Hex:fucoxanthin (fuc) ratio for type B/C was 11 times greater than that for type A, and the proportion of fuc in type A was 6 times higher than that in type B/C. The fuc derivative 4‐keto‐19′‐hexanoyloxyfucoxanthin (4‐keto‐hex) was present in type A but undetected in B/C. DNA sequencing of tufA distinguished morphotypes A, B/C (indistinguishable from B), and R, while little variation was observed within morphotypes. Thirty single nucleotide polymorphisms were identified in the 710 bp tufA sequence, of which 10 alleles were unique to B/C and B morphotypes, seven alleles were unique to type A, and six alleles were unique to type R. We propose that the morphologically, physiologically, and genetically distinct Southern Ocean type B/C sensu Young et al. (2003) be classified as E. huxleyi var. aurorae var. nov. S. S. Cook et Hallegr.     

Immunotoxins up to the present day

Immunotoxins consist of monoclonal or polyclonal antibodies conjugated to bacterial or plant toxins. The toxins used are typically of the A-B type in which a toxic A chain is coupled to a B chain responsible for cell binding and facilitation of A chain entry into the cytosol. Two broad strategies have been followed: coupling intact toxins, or A chains alone, to antibodies. This review examines current progress inin vitro andin vivo research, including recent clinical studies, concentrating principally on ricin or ricin A chain conjugates. The future role of conjugates using membrane-acting toxins, immunolysins, is also discussed.     

Bacillus sphaericus Mtx1 and Mtx2 toxins co-expressed in Escherichia coli are synergistic against Aedes aegypti larvae

Mtx1 and Mtx2 are mosquitocidal toxins produced by some strains of Bacillus sphaericus during vegetative phase of growth. Mtx1 from B. sphaericus 2297 shows higher toxicity against Culex quinquefasciatus larvae than to Aedes aegypti larvae whereas Mtx2 from B. sphaericus 2297 shows lower toxicity against C. quinquefasciatus than to A. aegypti larvae. To test synergism of these toxins against A. aegypti larvae, mtx1 and mtx2 genes were cloned into a single plasmid and expressed in Escherichia coli. Cells producing both Mtx1 and Mtx2 toxins exhibited high synergistic activity against A. aegypti larvae approximately 10 times compared to cells expressing only a single toxin. Co-expression of both toxins offers an alternative to improve efficacy of recombinant bacterial insecticides. There is a high possibility to develop these toxins to be used as an environmentally friendly mosquito control agent.     

Purification and characterization of the cytotoxic Cerebratulus A toxins

Mucus secreted from the skin of a marine worm, Cerebratulus lacteus, contains a family of polypeptide cytotoxins (A toxins) in addition to the previously reported polypeptide neurotoxins (B toxins). The A toxins were purified by Sephadex G-50 chromatography and then CM-cellulose gradient chromatography at pH 7.5 and pH 3.5. The three most abundant A toxins (designated according to their order of CM-cellulose elution) were homogeneous by gel electrophoreses, amino acid composition, and by NH2-terminal and COOH-terminal partial sequence analyses. Each of the three A toxins consists of a single basic polypeptide chain of 93 to 99 residues, cross-linked by three or four disulfide bonds, lacking reducing sugar and cysteinyl residues. The three A toxins rapidly lysed human red cells and Ehrlich ascites tumor cells at 1 to 10 microgram/ml concentrations. On a molar basis toxin A-III is about 4 times more active than melittin (bee venom lysin) and over 10 times more active than cardiotoxin (elapid snake lysin) upon human red cells. Purified A toxins lacked phospholipase A activity. The cytoxins as well as the neurotoxins were concentrated within the body wall integument.     

Synergism of mosquitocidal toxicity between CytA and CrylVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio-assays against first instars of Aedes aegypti. The LC₅₀ for the CytA inclusion was 60 ng ml⁻¹, whereas the LC₅₀ for the CryIVD was 85ng ml⁻¹ In comparison, the LC₅₀s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml⁻¹, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.     

Importance of cecum inClostridium botulinum colonization of mice and relationship of organism to large bowel

Germ-free mice, tested more than two weeks after removing their cecum, were at least 10⁴ times more resistant than controls to intestinal colonization byClostridium botulinum type A or B. Histologic examinations were done with the cecum and colon taken fromC. botulinum-monoassociated mice. Specimens from mice with severe botulism had normal appearance. In specimens from chronically monoassociated mice, the pathogen was in the lumen, but not attached to the intestine. Scanning electron microscopy suggested a possible association of the pathogen with the mucous gel lining.     

Botulinum Toxins Inhibit the Antidiuretic Hormone (ADH)-Stimulated Increase in Rabbit Cortical Collecting-Tubule Water Permeability

The mammalian renal collecting duct increases its water permeability in response to antidiuretic hormone (ADH). ADH causes cytoplasmic endosomes containing the water channel, aquaporin 2 (AQP2), to fuse with the apical membrane so that the water permeability of the tubule increases many times above baseline. SNARE proteins are involved in the docking and fusion of vesicles with the cell membrane in neuron synapses. Whether these proteins are involved in the fusion of vesicles to the cell membrane in other tissues is not entirely clear. In the present study, we examined the role of SNARE proteins in the insertion of water channels in the collecting-duct response to ADH by using botulinum toxins A, B and C. Toxins isolated from clostridium botulinum are specific proteases that cleave different SNARE proteins and inactivate them. Tubules were perfused in vitro with botulinum toxin in the perfusate (50 nM for A and B and 15 nM for C). ADH (200 pM) was then added to the bath after baseline measurements of osmotic water permeability (P_f) and the change in P_f was followed for one hour. Botulinum toxins significantly inhibited the maximum P_f by approximately 50%. Botulinum toxins A and C also decreased the rate of rise of P_f. Thus, SNARE proteins are involved in the insertion of the water channels in the collecting duct.     

Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH)

The development of the gut is controlled and modulated by different interacting mechanisms such as, genetic endowment, intrinsic biological regulatory functions, environment influences and last but no least, the diet influence. Considered together with other endogenous and exogenous factors the type of feeding may interfere greatly in the regulation of the intestinal microbiota. During the last years molecular methods offer a complementarity to the classic culture-based knowledge. FISH has been applied for molecular evaluation of the microbiota in newborns delivered by vaginal delivery. Eleven probes/probe combinations for specific groups of faecal bacteria were used to determine the bacterial composition in faecal samples of newborns infants under different types of feeding. Breast-fed infants harbor a fecal microbiota by more than two times increased in numbers of Bifidobacterium cells when compared to formula-fed infants. After formula-feeding, Atopobium was found in significant counts and the numbers of Bifidobacterium dropped followed by increasing numbers in Bacteroides population. Moreover, under formula feeding the infants microbiota was more diverse.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

A Possible Murine Model for Investigation of Pathogenesis of Sudden Infant Death Syndrome

Several studies have indicated a possible causative role of toxigenic bacteria in sudden infant death syndrome (SIDS). This study examined the effect of toxigenic E. coli on pregnant and infant mice to determine if these animals could be used as a model for SIDS pathogenesis. Strains of E. coli from the intestinal contents of infants who have died of SIDS or other causes and from the faeces of healthy infants were collected over a broad time scale. The isolates were tested for their ability to produce then known toxins of E. coli and were serotyped (O and H antigens). Certain serotypes (e.g. O1:H- and O25:H1) emerged significantly more frequently from cases of SIDS than from healthy infants and isolates of these types were generally toxigenic in Vero-cell cultures but whose verotoxicity was not related to classical Shiga or other known toxins. This mouse model was developed to test the effects of these toxigenic and also non-toxigenic strains. Four apparently healthy pups aged between 17 and 21 days died unobserved overnight but no pups of the 54 control mice died suddenly (P = 0.0247, Fisher’s exact test). These were considered to represent sudden unexpected deaths. Pathological effects compatible with those in SIDS were observed in mouse pups exposed to toxigenic strains indicating this model may be suitable for further study into the pathogenesis of unexpected deaths in infancy. Providing an animal model of SIDS would promote a much better avenue for studying the pathogenesis of this enigmatic condition.     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

Evaluation of the toxicity of eggs,hatchlings and tadpoles of the introduced toad Bufo marinus (Anura: Bufonidae) to native Australian aquatic predators

Abstract The early life history stages of anurans in the Family Bufonidae often possess chemicals that are noxious or toxic to predators. Predators with no evolutionary history of exposure to bufomds may be particularly susceptible to these toxins. We conducted a series of laboratory experiments to investigate the toxic effects of eggs, hatchlings and tadpoles of the introduced toad, Bufo marinus (Linnaeus), on native Australian aquatic predators. There was considerable interspecific and intraspecific variation in these effects. Bufo marinus were highly toxic to some predator species, but were readily consumed by other species without apparent ill effect. Interspecific variation in toxic effects was not related to predator feeding mode or the number of B. marinus ingested by predators, and there was no clear pattern of distribution of vulnerability among species within higher taxa. Intraspecific variation in responses to toxins may result from individual variation in the resistance of predators to B. marinus toxins, or from individual variation in toxicity among B. marinus. Some native species adversely affected by B. marinus appeared unable to detect and avoid B. marinus toxins. This may result from a general inability to assess the toxicity of food items or from a lack of evolutionary exposure to B. marinus toxins.     

Response of larval Chironomus tepperi (Diptera: Chironomidae) to individual Bacillus thuringiensis var. israelensis toxins and toxin mixtures

The biopesticide Bacillus thuringiensis israelensis (B.t.i.) is highly toxic to the larvae of Chironomus tepperi, an important pest of aerially sown rice in southern Australia. In this study, all of the known Cry genes and the Cyt1A gene from B.t.i. were expressed and tested for individual toxicity against fourth instar C. tepperi larvae. Possible synergism between toxins in two component mixtures involving all toxins except Cry10A was also evaluated. Of the Cry toxins, only Cry11A and Cry4B displayed substantial toxicity; however, both were 10- to 20-fold less toxic than the parental B.t.i. strain. The only detected synergy was between the mildly toxic Cry4A and Cyt1A toxins. In direct contrast to previous studies with mosquitoes, mixtures of Cry11A/Cry4B and Cry11A/Cyt1A were mildly antagonistic. The activity of Cry11A and Cry4B is sufficient to justify investigation as to whether their expression in transgenic rice plants could provide control of C. tepperi larvae.     

Uptake and localization of fluorescently‐labeled Karenia brevis metabolites in non‐toxic marine microbial taxa

Brevetoxin (PbTx) is a neurotoxic secondary metabolite of the dinoflagellate Karenia brevis. We used a novel, fluorescent BODIPY‐labeled conjugate of brevetoxin congener PbTx‐2 (B‐PbTx) to track absorption of the metabolite into a variety of marine microbes. The labeled toxin was taken up and brightly fluoresced in lipid‐rich regions of several marine microbes including diatoms and coccolithophores. The microzooplankton (20–200 μm) tintinnid ciliate Favella sp. and the rotifer Brachionus rotundiformis also took up B‐PbTx. Uptake and intracellular fluorescence of B‐PbTx was weak or undetectable in phytoplankton species representative of dinoflagellates, cryptophytes, and cyanobacteria over the same (4 h) time course. The cellular fate of two additional BODIPY‐conjugated K. brevis associated secondary metabolites, brevenal (B‐Bn) and brevisin (B‐Bs), were examined in all the species tested. All taxa exhibited minimal or undetectable fluorescence when exposed to the former conjugate, while most brightly fluoresced when treated with the latter. This is the first study to observe the uptake of fluorescently‐tagged brevetoxin conjugates in non‐toxic phytoplankton and zooplankton taxa, demonstrating their potential in investigating whether marine microbes can serve as a significant biological sink for algal toxins. The highly variable uptake of B‐PbTx observed among taxa suggests some may play a more significant role than others in vectoring lipophilic toxins in the marine environment.     

Activation of Clostridium botulinum type B and E derivative toxins with lysine-specific proteases

Abstract Clostridium botulinum type B and E derivative toxins were activated with lysyl endopeptidase or endoproteinase Lys-C, which splits only the bond involving the carboxyl group of a lysine residue. Type B toxin was more efficiently activated with lysyl endopeptidase; type E toxin was more efficiently activated with trypsin. Type B toxin was split by the lysine-specific protease into 2 fragments of molecular sizes indistinguishable from those induced with trypsin. Type E toxin was split by the same protease into 3 fragments, 2 of which had M _r identical to those obtained with trypsin, the other having an M _r less than that of the heavy chain but greater than that of the light chain. These results attest that both activation and nicking of type B and E derivative toxins are ascribable to cleavage, not of an arginyl, but of a lysyl bond.     

Activity spectra of Bacillus Thuringiensis δ-endotoxins against eight insect cell lines

Summary Eight continuous insect cell lines were tested for susceptibility to the δ-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins. IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains. The in vitro assay system, referred to as the “lawn assay” and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived.