The haemolysins produced by Aeromonas species were detected and compared by two assay methods--a modified blood agar plate assay and the rabbit erythrocyte haemolysin method. Both assays showed a high level of agreement (86%). The titres of the rabbit erythrocyte haemolysin assay correlated with the haemolytic zone diameter of the ox blood agar assay. In addition the agar haemolysin assay had simple media requirements, was easy to perform and results were well defined.
A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
Forty clinical isolates of Vibrio parahaemolyticus were studied for the production of the thermostable direct hemolysin (TDH), and the TDH-related hemolysin (TRH) including the respective encoding genes, tdh and trh. The presence of TDH and its encoding genes were found amongst 95% of the strains, whereas the TRH was absent amongst these isolates. Thirty-two isolates were found to be plasmid-free, whereas eight isolates possessed plasmids with sizes ranging from 2.4 > or = 23 kb. Using a DNA probe coding for the homologous region of the tdh and trh, it was found that the tdh genes were present on the chromosomal DNA.
A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.
Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.
Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F(4) to F(8)) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F(9) found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F(9). The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F(9) to F(13)) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with (125)I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F(15)). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F(2) progeny from a backcross of F(1) progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.
Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.
A field population of Plutella xylostella from Malaysia (SERD4) was divided into five sub-populations and four were selected (G2-G5) with the Bacillus thuringiensis insecticidal crystal (Cry) toxins Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da. Bioassay at G6 gave resistance ratios of 88, 5, 2 and 3 for Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da respectively compared with the unselected sub-population (UNSEL-SERD4). The Cry1Ac-selected population showed little cross-resistance to Cry1Ab, Cry1Ca and Cry1Da, (3-, 2- and 3-fold compared with UNSEL-SERD4), whereas the Cry1Ab-SEL sub-population showed marked cross-resistance to Cry1Ac (40-fold), much greater than Cry1Ab itself. In contrast, the Cry1Ca- and Cry1Da-SEL sub-population showed little if any cross-resistance to Cry1Ac and Cry1Ab. The mode of inheritance of resistance to Cry1Ac was examined in Cry1Ac-selected SERD4 by standard reciprocal crosses and back-crosses using a laboratory insecticide-susceptible population (ROTH). Logit regression analysis of F1 reciprocal crosses indicated that resistance to Cry1Ac was inherited as an incompletely dominant trait. At the highest dose of Cry1Ac tested, resistance was recessive, while at the lowest dose it was almost completely dominant. The F2 progeny from a back-cross of F1 progeny with ROTH were tested with a concentration of Cry1Ac that would kill 100% of ROTH. The mortality ranged between 50 and 95% in seven families of back-cross progeny, which indicated that more than one allele on separate loci were responsible for resistance to Cry1Ac.
Resistance to the bacteria-derived insecticides spinosad (Conserve), abamectin (Vertimec), Bacillus thuringiensis var kurstaki (Btk) (Dipel), B thuringiensis var aizawai (Bta) (Xentari), B thuringiensis crystal endotoxins Cry1Ac and Cry1Ca, and to the synthetic insecticide fipronil was estimated in a freshly-collected field population (CH1 strain) of Plutella xylostella (L) from the Cameron Highlands, Malaysia. Laboratory bioassays at G1 indicated significant levels of resistance to spinosad, abamectin, Cry1Ac, Btk, Cry1Ca, fipronil and Bta when compared with a laboratory insecticide-susceptible population. Logit regression analysis of F1 reciprocal crosses indicated that resistance to spinosad in the CH1 population was inherited as a co-dominant trait. At the highest dose of spinosad tested, resistance was close to completely recessive, while at the lowest dose it was incompletely dominant. A direct test of monogenic inheritance based on a back-cross of F1 progeny with CH1 suggested that resistance to spinosad was controlled by a single locus.
Bioassays (at generation 1, G1) using fipronil, spinosad, indoxacarb, and Bacillus thuringiensis toxins Cry1Ac and Cry1Ca with a newly collected field population of Plutella xylostella (L.) from farmers fields in the Cameron Highlands, Malaysia, indicated a resistance ratio of approximately 400-, 1,170-, 330-, 2,840-, and 1,410-fold, respectively, compared with a laboratory-susceptible population of P. xylostella (ROTH). At G3, the field-derived population was divided into two subpopulations, one was selected (G3 to G7) with fipronil (fip-SEL), whereas the second was left unselected (UNSEL). Bioassays at G8 found that selection with fipronil gave a resistance ratio of approximately 490 compared with UNSEL and approximately 770 compared with ROTH. The resistance ratio for fipronil, spinosad, indoxacarb, Cry1Ac, and Cry1Ca in the UNSEL population declined significantly by G8. Logit regression analysis of F1 reciprocal crosses between fip-SEL (at G8) and UNSEL indicated that resistance to fipronil in the fip-SEL population was inherited as an autosomal, incompletely recessive (D(LC) = 0.37) trait. At the highest dose of fipronil tested, resistance was completely recessive, whereas at the lowest dose it was incompletely recessive. A direct test of monogenic inheritance based on a backcross of F1 progeny with fip-SEL suggested that resistance to fipronil was controlled by a single locus. The fip-SEL population at G8 showed little change in its response to spinosad and indoxacarb compared with G1, whereas its susceptibility to Cry1Ac and Cry1Ca increased markedly over the selection period. This suggests that there may be some low level of cross-resistance between fipronil, spinosad, and indoxacarb.
The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.
This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption.
We constructed recombinant phage particles displaying the Bacillus thuringiensis Cry1Ba4 active toxin using the pfUSE5 and pComb3X phagemid vectors. The recombinant phage particles were screened and evaluated for displayed biologically active Cry1Ba4 toxin against the target insect larvae. Concurrent expression of Cry1Ba4 protoxin was carried out using the pETBlue -2 plasmid expression vector in Escherichia coli Tuner (DE3)pLacI and the protoxin was successfully expressed at a size of 129 kDa. In the bioassay, 3.30 mg crude extract of Cry1Ba4 protoxin, 9.35 x 10(9) TU and 7.70 x 10(9) TU of induced recombinant phage particles carrying Cry1Ba4 active toxin displayed on pComb3X and pFUSE5, respectively, demonstrated mortality of greater than 85% against Plutella xylostella (third-instar) within 48 hours. Thus, we have successfully displayed the Cry1Ba4 activated toxin on the surface of a phage and demonstrated toxicity towards larvae.
Polymerase chain reaction (PCR) technique was used to assay for the detection of specific genes in the genomes of the Aeromonas spp. isolated from environmental and shellfish sources, particularly aero and hlyA genes, responsible for aerolysin and hemolysin toxins production in this genus. The results showed that: (i) the 1500 bp amplicon of the hlyA gene was detected in 20/38 of the Aeromonas hydrophila, 13/38 of the A. caviae and 6/9 of the A. veronii biovar sobria isolates; (ii) the 690 bp amplicon of the aero gene was detected in 20/38 of A. hydrophila, 17/38 of A. caviae and 6/9 of A. veronii biovar sobria isolates; (iii) the nucleotide blast results of aerolysin gene sequences of the representative strains of A. hydrophila, A. caviae and A. veronii biovar sobria revealed a high homology of 94%, 95% and 95% with published sequences, respectively and ; (iv) the protein blast showed 97%, 94% and 96% homology when compared to the published sequences, respectively. The finding of A. hydrophila virulence genes in other members of the genus Aeromonas, may give a new perspective to the significance of these results. The method described here may be a useful detection tool to assist in further investigation of aero and hlyA genes in the genus Aeromonas, especially for food microbiologist.
Bacillus thuringiensis Berliner (Bt) crystal (Cry) toxins are expressed in various transgenic crops and are also used as sprays in integrated pest management and organic agricultural systems. The diamondback moth (Plutella xylostella L.) is a major worldwide pest of crucifer crops and one that has readily acquired field resistance to a broad range of insecticides.
A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 x 10(4) cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.