Bt Infection Research Articles

Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis

ABSTRACTMicroevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susceptible insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management and immune defense in the fat body. Following oral infection with Bt, the expression of these genes was further elevated in the fat body and midgut of both lines and to a greater extent some of them in resistant line than the susceptible line. This gene expression analysis reveals a pattern of resistance mechanisms targeted to sites damaged by Bt with the insect placing greater emphasis on tissue repair as revealed by elevated expression of these genes in both the fat body and midgut epithelium. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affects the viability of other gut bacteria. Remarkably the resistant line employs multifactorial adaptations for resistance to Bt without any detected negative trade off since the insects exhibited higher fecundity.

Seroprevalence of bluetongue in ruminants of Jharkhand.

Aim:This study was carried out to assess the presence of anti-bluetongue (BT) antibodies in sheep, goat and cattle of different agro-climatic zones of Jharkhand.Materials and Methods:Serum samples were collected from apparently healthy as well as suspected sheep, goat and cattle from different districts of Jharkhand covering different agro-climatic zones. Serum samples were screened by indirect enzyme linked immunosorbent assay (iELISA) for detecting anti-BT antibodies.Results:Out of a total of 480 animal serum samples (sheep-190, goats-210 and cattle-80) screened, 83 (43.68%) of sheep, 91 (43.33%) of goat and 46 (57.50%) of cattle sera were found positive. The % positivity ranged between 41% and 51% in different agro-climatic zones. The results showed slight higher seroprevalence, although not significantly, in cattle than sheep and goats in different agro-climatic zones of Jharkhand.Conclusions:The above data indicate widespread prevalence of BT virus antibodies in studied areas. The incidence of BT is not detected officially, so far. The present seroprevalence status of BT in Jharkhand indicates presence of BT infection in the state for the first time.

Trans‐generational priming of resistance in wild flour beetles reflects the primed phenotypes of laboratory populations and is inhibited by co‐infection with a common parasite

Summary Trans‐generational priming renders offspring of immune‐challenged parents less susceptible to disease‐induced mortality, and has been demonstrated in a variety of arthropod taxa under controlled laboratory conditions. However, relatively little is known about the applicability of these laboratory results to priming in wild populations, especially in the context of environmental variables like maternal co‐infection and potential trade‐offs with other life history traits. We performed nearly parallel trans‐generational priming experiments on laboratory and wild strains of Tribolium beetles against the bacterial pathogen Bacillus thuringiensis (Bt). We investigated the impact of maternal immune challenge on offspring survival and development time, as well as the correlation between development and survival by priming status. Furthermore, we manipulated wild beetle parental co‐infection with a gregarine parasite that was prevalent in the wild population in order to determine the impact of co‐infection on the efficacy of trans‐generational priming. Trans‐generational priming resulted in a large but birth order‐dependent acceleration of offspring development time, and enhanced offspring survival against infection, in both laboratory and wild populations. However, adult parental co‐infection with gut protozoa severely curtailed primed offspring survival against Bt infection. These results suggest that shifts in life history traits associated with trans‐generational priming in laboratory model systems extend to wild populations. These traits, including survival, development, and interference from co‐infection, are important parameters for predicting the impact of priming on disease dynamics in the wild.

Alteration in Bacillus thuringiensis toxicity by curing gut flora: novel approach for mosquito resistance management

Mosquitoes are known for acquiring resistance against insecticides in many ways, namely target side mutation, enzyme modification, sequestration, quick elimination, etc. But, the role of microflora present in abundance in the larval midgut is less explored with respect to their role in insecticide resistance. During the course of their development, mosquitoes are continuously exposed to microbes and have naturally acquired midgut microbial flora. This midgut flora can modulate the mosquito's susceptibility to Bacillus thuringiensis (Bt) infection by degrading toxic Bt protein forms through an unknown mechanism. In this study, we show that microbe-free aseptic mosquito larvae displayed an increased susceptibility to Bt toxicity compared to larvae harboring natural microbial flora. Fourth instar larvae of Anopheles stephensi were treated separately with penicillin, streptomycin, erythromycin (100μg/ml), and mixtures of all three antibiotics and then analyzed for Bt toxicity. We have also examined the influence of the mosquito's midgut microbial flora under microaerophilic condition on the Bt protein degradation through plate, broth, TLC, and UV-vis spectrophotometric assay. A better understanding of the roles of microbiota in preventing Bt toxicity to mosquitoes could potentially lead to the development of new sustainable mosquito control strategies.

Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

Synergistic effects of Cydia pomonella granulovirus GP37 on the infectivity of nucleopolyhedroviruses and the lethality of Bacillus thuringiensis

To initiate an efficient primary infection, it is important for baculovirus virions to penetrate through the peritrophic membrane (PM) of the host insect. It is frequently reported that enhancins of baculoviruses significantly enhance viral infection by degrading the various protein components of PMs. However, not all baculoviruses encode enhancins. GP37s of baculoviruses share high amino acid identity with fusolins, synergistic factors found in entomopoxviruses. In this study, a truncated Cydia pomonella granulovirus GP37 was expressed in Escherichia coli. The expressed GP37 effectively bound to chitin, and binding occurred predominantly within 3h. GP37 altered the protein profiles of Spodoptera exigua PMs, from which a 50-kDa protein was dissociated. Droplet-feeding bioassays indicated that GP37 significantly enhanced the infectivity of nucleopolyhedroviruses (NPVs) and the lethality of Bacillus thuringiensis (Bt) in S. exigua larvae. This is the first demonstration of the enhancement of NPVs and Bt infection by a baculovirus GP37.

Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae)

Bacillus thuringiensis is one of the most widely used sources of biorational pesticides, as well as a key source of genes for transgenic expression to provide pest resistance in plants. In this study the effect of Bacillus thuringiensis ssp. galleriae (Bt) infection on the activity of superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), concentrations of oxidated and reduced thiols (RSSR/RSH) and malondialdehyde (MDA) was tested in the midgut of Galleria mellonella larvae. We found that Bt infection resulted in increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio the first day after inoculation. However, catalase activity decreased on the first and following days after bacterial infection by Bt. Our results confirm the hypothesis that Bt infection increases the level of oxidative stress in the larval midgut. In light of this study, it seems possible that oxidative damage contributes to cell death in the midgut during bacteriosis.

Potentiating Effect of <I>Bacillus thuringiensis</I> subsp. <I>kurstaki</I> on Pathogenicity of Entomopathogenic Bacterium <I>Xenorhabdus nematophila</I> K1 Against Diamondback Moth (Lepidoptera: Plutellidae)

Xenorhabdus nematophila is the symbiotic bacterium of an entomopathogenic nematode, Steinernema carpocapsae. When the nematode enters a target insect, the symbiotic bacteria are released into the hemocoel. After inducing host immunosuppression, the bacteria multiply in the hemocoel and cause fatal septicemia. For optimal field application to control insect pests, culturing mass numbers of the nematodes would be costly. In this study, Bacillus thuringiensis (Bt) was chosen as an alternative natural vector, which would be relatively economical for field application. Bt infection of gut epithelium would form a bacterial passage between the gut lumen and hemocoel, which facilitates the orally fed X. nematophila to infect the hemocoel. Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), used in this study was tolerant to Bt because only 10% mortality was noted in response to 2 times higher concentration than recommended for commercial B. t. kurstaki, although this species was susceptible only during early instars. The orally fed X. nematophila caused significant mortality to early instars of P. xylostella, but not late instars. When both X. nematophila and Bt were fed to late instars of P. xylostella, they showed significantly enhanced mortality, in which X. nematophila cells were recovered from the hemocoel of the treated P. xylostella. However, when only X. nematophila was fed, no cells were recovered from the hemolymph. This study suggests that X. nematophila can be applied to control P. xylostella in a mixture with Bt in the field without its nematode host.

Sublethal Effects ofBacillus thuringiensisBerliner subsp.kurstakionLymantria dispar(Lepidoptera: Lymantriidae) and the Tachinid ParasitoidCompsilura concinnata(Diptera: Tachinidae)

Parasitoid-pathogen interactions were examined using gypsy moth, Lymantria dispar (L.), Bacillus thuringiensis Berliner, and Compsilura concinnata (Meigen). The objectives of this study were to quantify effects of sublethal doses of Bacillus thuringiensis (Bt) force-fed to gypsy moths and to determine if sublethal doses of Bt affected host acceptance and suitability of gypsy moth for C. concinnata. Gypsy moths were minimally affected by sublethal doses of Bt; development of fourth instar was delayed, and male pupal mass reduced. Compsilura concinnata preferentially attacked and had higher superparasitism on noninfected hosts than on Bt-treated larvae. Exposure of gypsy moth to both sublethal doses of Bt and parasitoids reduced percentage parasitism and host larval survivorship. Effects on C. concinnata development varied with host superparasitism status. Parasitoids in Bt-treated, superparasitized gypsy moths had shorter larval development times and smaller pupal masses than parasitoids in untreated larvae, while parasitoids in singly parasitized larvae had larger pupal masses than those in superparasitized larvae. Timing of Bt infection relative to parasitism is a factor in gypsy moth mortality, but not in parasitoid potential fecundity.

The mammalian safety of Bacillus thuringiensis-based insecticides.

The United States Environmental Protection Agency between the years 1961 and 1995 registered 177 products containing viable Bacillus thuringiensis (Bt). Numerous laboratory studies have demonstrated that Bt and Bt products are noninfectious and are toxic to mammals only at a dose > or =10(8) colony forming units (cfu) per mouse (a human equivalent based on the weight of >10(11) cfu). In contrast, as few as three vegetative cells of Bacillus anthracis can kill mice (a human equivalent of >10(3) cfu). There are only two literature reports of Bt infection in man between the year 1997 and the present, and all infected individuals had experienced either extensive burns or a blast injury, which predisposed them to infection. Two epidemiology studies conducted during large-scale aerial Bt serovar kurstaki spray campaigns reported no increased incidence of illness. Some recent papers have expressed concern about the production of Bacillus cereus enterotoxins by Bt isolates. Laboratory studies found no evidence of illness in rats and sheep fed Bt products, nor have epidemiology studies found increased incidence of diarrhea during Bt aerial spray campaigns. Increases in human antibody levels following exposure to Bt products have been reported but there was no increased incidence in asthma or other illness. Based on laboratory studies and field experience, Bt insecticides have an excellent safety record.

Bluetongue: Laboratory diagnosis

Definitive diagnosis of bluetongue virus (BTV) infection, often subclinical in domestic and wild ruminant relies heavily on laboratory techniques for BTV isolation and demonstration of BTV antigens, viral nucleic acids and antibodies. The virus can be isolated from blood components, mainly the erythrocyte fraction, collected from affected animals during the period of febrile response. Semen collected from male animals at the peak of viremia and tissues from affected animals and fetuses may also be used for BTV isolation. The primary procedure for BTV isolation is inoculation of embryonated chicken eggs with a subpassage onto cell cultures (e.g. BKH-21, Vero cell lines). In addition to the conventional techniques such as fluorescent antibody staining and virus neutralization procedures for sero-grouping and serotyping of BTV isolates, immunohistochemical, immunoenzymatic and immunoelectron microscopic techniques, using monoclonal antibodies (MAb), offer more rapid, specific and sensitive approaches for BTV identification and antigen detection. The progress of molecular biology, especially the development of genetic probes for hybridization analysis and polymerase chain reaction techniques for detection of BTV nucleic acids hold the promise of most efficient diagnostic assays. Among the various serogroup-specific assays for antibody detection, the agar gel immunodiffusion (AGID) and competitive (C) ELISA are the most widely used tests. Because of its limitations (i.e. anticomplementary serum and complexity of the procedure) the complement fixation (CF) test is virtually abandoned and is used in only a few laboratories. Although the AGID test is simple to perform and rapid, it is not highly sensitive or quantitative and has limitations in its specificity. Sera containing antibodies to other group of Orbiviruses (e.g. epizootic hemorrhagic disease) may result in non-specific reaction in the AGID test. Among several ELISAs that have recently been developed, the C.ELISA in which a group-specific MAb to BTV is used, has proved to be the most sensitive and specific assay for detection of antibodies to BTV. Following extensive national and international validation, the C.ELISA is gradually replacing the AGID as a universal test to certify ruminants for trade purposes and to diagnose BT infection in domestic and wild animals. The cell culture-based microtiter serum neutralization (MTSN) is the most commonly used assay for the detection of serotype-specific antibodies to the recognized BTVs in animal sera. The MTSN may be used to type virus isolates and also to monitor animal population for specific serotypes of BTV in epidemiological investigations.