Development and laboratory validation of a plant-derived repellent blend, effective against Aedes aegypti [Diptera: Culicidae], Anopheles gambiae [Diptera: Culicidae] and Culex quinquefasciatus [Diptera: Culicidae].

Mosquitoes of the genera Aedes, Anopheles and Culex vector a wide range of pathogens seriously affecting humans and livestock on a global scale. Over-reliance on insecticides and repellents has driven research into alternative, naturally-derived compounds to fulfil the same objectives. Steam distilled extracts of four plants with strong, yet attractive, volatile profiles were initially assessed for repellency in a dual-port olfactometer using Aedes aegypti as the model species. Picea sitchensis was found to be the most repellent, proving comparable to leading products when applied at 100% (p = 1.000). Key components of conifer-derived volatile profiles were then screened via electroantennography before those components eliciting an electrophysiological response were assayed individually in the olfactometer; according to WHO protocol. The most promising 5 were selected for reductive analyses to produce an optimised semiochemical blend. This combination, and a further two variations of the blend, were then progressed to a multi-species analysis using the BG-test whereby bite-attempt frequency on hands was assessed under different repellent treatments; assays were compared between Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Efficacy was found against all three species, although it was found that Ae. aegypti was the most susceptible to the repellent, with An. gambiae being the least. Here, a novel, naturally-derived blend is presented with weak spatial repellency, as confirmed in laboratory assays. Further work will be required to assess the full extent of the potential of the products, both in terms of field application and species screening; however, the success of the products developed demonstrate that plant metabolites have great capacity for use in the repellent sector; both to improve upon known compounds and to reduce the usage of toxic products currently on the market.

Identification of the odorant binding proteins of Western Flower Thrips (Frankliniella occidentalis), characterization and binding analysis of FoccOBP3 with molecular modelling, molecular dynamics simulations and a confirmatory field trial.

Olfactory systems are indispensable for insects as they, including Western Flower Thrips (Frankliniella occidentalis), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis. Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3. Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3. The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

Evaluation of Fungal Volatile Organic Compounds for Control the Plant Parasitic Nematode Meloidogyne incognita.

Plant parasitic nematodes are a serious threat to crop production worldwide and their control is extremely challenging. Fungal volatile organic compounds (VOCs) provide an ecofriendly alternative to synthetic nematicides, many of which have been withdrawn due to the risks they pose to humans and the environment. This study investigated the biocidal properties of two fungal VOCs, 1-Octen-3-ol and 3-Octanone, against the widespread root-knot nematode Meloidogyne incognita. Both VOCs proved to be highly toxic to the infective second-stage juveniles (J2) and inhibited hatching. Toxicity was dependent on the dose and period of exposure. The LD50 of 1-Octen-3-ol and 3-Octanone was 3.2 and 4.6 µL, respectively. The LT50 of 1-Octen-3-ol and 3-Octanone was 71.2 and 147.1 min, respectively. Both VOCs were highly toxic but 1-Octen-3-ol was more effective than 3-Octanone. Exposure of M. incognita egg-masses for 48 h at two doses (0.8 and 3.2 µL) of these VOCs showed that 1-Octen-3-ol had significantly greater nematicidal activity (100%) than 3-Octanone (14.7%) and the nematicide metham sodium (6.1%). High levels of reactive oxygen species detected in J2 exposed to 1-Octen-3-ol and 3-Octanone suggest oxidative stress was one factor contributing to mortality and needs to be investigated further.

Entomopathogenic Fungi in Biological Plant Protection: The Machinery of Multicomponent System Interactions.

Plant protection faces a growing number of challenges, partly stemming from intensification of plant cultivation to ensure food security for a rapidly growing global population [...].

The Potential of Metarhizium anisopliae Blastospores to Control Aedes aegypti Larvae in the Field.

Entomopathogenic fungi are promising as an environmentally benign alternative to chemical pesticides for mosquito control. The current study investigated the virulence of Metarhizium anisopliae blastospores against Aedes aegypti under both laboratory and field conditions. Virulence bioassays of conidia and blastospores were conducted in the laboratory, while field simulation bioassays were conducted under two conditions: totally shaded (TS) or partially shaded (PS). In the first bioassay (zero h), the larvae were added to the cups shortly after the preparation of the blastospores, and in the subsequent assays, larvae were added to the cups 3, 6, 9, and 12 days later. The survival of the larvae exposed to blastospores in the laboratory was zero on day two, as was the case for the larvae exposed to conidia on the sixth day. Under TS conditions, zero survival was seen on the third day of the bioassay. Under PS conditions, low survival rates were recorded on day 7. For the persistence bioassay under PS conditions, low survival rates were also observed. Metarhizium anisopliae blastospores were more virulent to Ae. aegypti larvae than conidia in the laboratory. Blastospores remained virulent under field simulation conditions. However, virulence rapidly declined from the third day of field bioassays. Formulating blastospores in vegetable oil could protect these propagules when applied under adverse conditions. This is the first time that blastospores have been tested against mosquito larvae under simulated field conditions, and the current study could be the basis for the development of a new biological control agent.

Evaluation of Metarhizium brunneum- and Metarhizium-Derived VOCs as Dual-Active Biostimulants and Pest Repellents in a Wireworm-Infested Potato Field.

Wireworm, the larval stages of click beetles, are a serious pest of tubers, brassicas and other important commercial crops throughout the northern hemisphere. No effective control agent has been developed specifically for them, and many of the pesticides marketed as having secondary application against them have been withdrawn from EU and Asian markets. Metarhizium brunneum, an effective entomopathogenic fungus, and its derived volatile metabolites are known to be effective plant biostimulants and plant protectants, although field efficacy has yet to be validated. Field validation of a combined M. brunneum and derived VOC treatments was conducted in Wales, UK, to assess the effects of each as a wireworm control agent and biostimulant. Plots were treated with Tri-Soil (Trichoderma atroviridae), M. brunneum, 1-octen-3-ol or 3-octanone, or combinations thereof. Treatments were applied subsurface during potato seeding (n = 52), and potatoes were harvested at the end of the growing season. Each potato was weighed individually and scored for levels of wireworm damage. Applications of both the VOCs and the M. brunneum individually were found to significantly decrease wireworm burden (p < 0.001). Combinations of M. brunneum and 3-octanone were also found to significantly decrease wireworm damage (p < 0.001), while no effect on yield was reported, resulting in an increased saleable mass over controls (p < 0.001). Herein, we present a novel 'stimulate and deter' wireworm control strategy that can be used to significantly enhance saleable potato yields and control wireworm populations, even under high pest pressure densities.

Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail.

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 - 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in "lure and kill" strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

On-farm Production of Microbial Entomopathogens for use in Agriculture: Brazil as a Case Study.

In Brazil, the production of beneficial microorganisms by growers exclusively for their own use is a practice known as "on-farm production". Regarding on-farm bioinsecticides, they were initially deployed for pests of perennial and semi-perennial crops in the 1970s but, since 2013, their use has extended to pests of annual crops such as maize, cotton, and soybean. Millions of hectares are currently being treated with these on-farm preparations. Local production reduces costs, meets local needs, and reduces inputs of environmentally damaging chemical pesticides, facilitating establishment of more sustainable agroecosystems. Critics argue that without implementation of stringent quality control measures there is the risk that the on-farm preparations: (1) are contaminated with microbes which may include human pathogens or (2) contain very little active ingredient, impacting on field efficacy. The on-farm fermentation of bacterial insecticides predominates, especially that of Bacillus thuringiensis targeting lepidopteran pests. However, there has been a rapid growth in the past 5 years in the production of entomopathogenic fungi, mostly for the control of sap-sucking insects such as whitefly (Bemisia tabaci (Gennadius)) and the corn leafhopper (Dalbulus maidis (DeLong and Wolcott)). In contrast, on-farm production of insect viruses has seen limited growth. Most of the ca. 5 million rural producers in Brazil own small or medium size properties and, although the vast majority still do not practice on-farm production of biopesticides, the topic has aroused interest among them. Many growers who adopt this practice usually use non-sterile containers as fermenters, resulting in poor-quality preparations, and cases of failure have been reported. On the other hand, some informal reports suggest on-farm preparations may be efficacious even when contaminated, what could be explained, at least partially, by the insecticidal secondary metabolites secreted by the pool of microorganisms in the liquid culture media. Indeed, there is insufficient information on efficacy and mode of action of these microbial biopesticides. It is usually the large farms, some with > 20,000 ha of continuous cultivated lands, that produce biopesticides with low levels of contamination, as many of them possess advanced production facilities and have access to specialized knowledge and trained staff. Uptake of on-farm biopesticides is expected to continue but the rate of adoption will depend on factors such as the selection of safe, virulent microbial strains and implementation of sound quality control measures (compliance with emerging Brazilian regulations and international standards). The challenges and opportunities of on-farm bioinsecticides are presented and discussed.

Drug Standardization through Pharmacognostic Approaches and Estimation of Anticancer Potential of Chamomile (Matricaria chamomilla L.) using Prostate-Cancer cell lines: An In-vitro Study.

Cancer is the major challenge across world and the adenocarcinoma of prostate malignancy is the second most prevalent male cancer. Various medicinal plants are used for the treatment and management of various cancers. Matricaria chamomilla L., is one of the extensively used Unani medicament for the treatment of various type of diseases. In the current study we evaluated most of the parameters prescribed for drug standardization using pharmacognostic approaches. The 2,2 Diphenyl-1-picryl hydrazyl (DPPH) method was utilized for the analysis of antioxidant activity in the flower extracts of M. chamomilla. Moreover, we analyzed the antioxidant and cytotoxic activity of M. chamomilla (Gul-e Babuna) through in-vitro method. DPPH (2,2-diphenyl-1-picryl-hydrazl-hydrate) method was utilized for the analysis of antioxidant activity in the flower extracts of M. chamomilla. CFU and wound healing assay were performed to determine the anti-cancer activity. The results demonstrated that various extracts of M. chamomilla fulfilled most of the parameters of drug standardization and contained good antioxidant and anticancer activities. The ethyl acetate showed higher anticancer activity followed by aqueous, hydroalcoholic, petroleum benzene and methanol by CFU method. Also, the wound healing assay demonstrated that ethyl acetate extract has more significant effect followed by methanol and petroleum benzene extract on prostate cancer cell line (C4-2). The current study concluded that the extract of M. chamomilla flowers could act as good source of natural anti-cancer compounds.

Dysregulation of Grainyhead-like 3 expression causes widespread developmental defects.

BACKGROUND: The gene encoding the transcription factor, Grainyhead-like 3 (Grhl3), plays critical roles in mammalian development and homeostasis. Grhl3-null embryos exhibit thoraco-lumbo-sacral spina bifida and soft-tissue syndactyly. Additional studies reveal that these embryos also exhibit an epidermal proliferation/differentiation imbalance. This manifests as skin barrier defects resulting in peri-natal lethality and defective wound repair. Despite these extensive analyses of Grhl3 loss-of-function models, the consequences of gain-of-function of this gene have been difficult to achieve. RESULTS: In this study, we generated a novel mouse model that expresses Grhl3 from a transgene integrated in the Rosa26 locus on an endogenous Grhl3-null background. Expression of the transgene rescues both the neurulation and skin barrier defects of the knockout mice, allowing survival into adulthood. Despite this, the mice are not normal, exhibiting a range of phenotypes attributable to dysregulated Grhl3 expression. In mice homozygous for the transgene, we observe a severe Shaker-Waltzer phenotype associated with hearing impairment. Micro-CT scanning of the inner ear revealed profound structural alterations underlying these phenotypes. In addition, these mice exhibit other developmental anomalies including hair loss, digit defects, and epidermal dysmorphogenesis. CONCLUSION: Taken together, these findings indicate that diverse developmental processes display low tolerance to dysregulation of Grhl3.

Identification of a Novel GRHL3/HOPX/Wnt/ß-Catenin Proto-oncogenic Axis in Squamous Cell Carcinoma of the Esophagus.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor long-term prognosis. The molecular mechanisms underlying the initiation and progression of this tumor are largely unknown. The transcription factor GRHL3 functions as a potent tumor suppressor in SCC of skin, head, and neck. This study aims to determine whether GRHL3 also plays a role in the homeostasis of the esophageal epithelium and in the development of ESCC. METHODS: The effects of Grhl3 deletion on squamous epithelial homeostasis in embryos and adult mice were examined using immunohistochemistry, transmission electron microscopy, and real-time polymerase chain reaction. The conditionally deleted mice were subsequently used to determine susceptibility to ESCC. Whole-transcriptome sequencing (RNA-seq) was performed on ESCC in wild-type and Grhl3 deleted animals. To decipher the signaling pathways, real-time polymerase chain reaction, immunohistochemistry, analysis of chromatin immunoprecipitation sequencing, chromatin immunoprecipitation-polymerase chain reaction, and RNA seq datasets were used. Primary human samples were used to validate the findings in the mouse model. RESULTS: Loss of Grhl3 perturbs the proliferation-differentiation balance in the esophageal epithelium, thereby increasing the susceptibility to esophageal carcinogenesis in adult mice. Grhl3 imparts its tumor suppressor function by regulating the expression of HOPX. We have identified the Wnt/ß-catenin pathway as the downstream effectors of GRHL3 and HOPX through our integrated approach using patient-derived ESCC samples and mouse models. CONCLUSIONS: GRHL3 conveys its tumor suppressor function in ESCC through regulating its target gene HOPX, which limits Wnt/ß-catenin signaling. Targeted therapies to inhibit this pathway could be a potential treatment strategy for ESCC patients with reduced GRHL3 expression.

Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor.

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Its Derived Volatile Organic Compounds as Biostimulants of Commercially Valuable Angiosperms and Gymnosperms.

Metarhizium brunneum is a highly effective entomopathogenic fungus that also functions as a plant biostimulant. It can act as both an endophyte and rhizosphere colonizer; however, the mechanisms driving biostimulation are multifactorial. In this work, oilseed rape (Brassica napus) seeds were grown in composts treated with different concentrations of M. brunneum strains ARSEF 4556 or V275, or the M. brunneum-derived volatile organic compounds 1-octen-3-ol and 3-octanone. Biostimulation efficacy was found to be strongly dose dependent. Concentrations of 1 × 106 conidia g-1 compost were found to be most effective for the M. brunneum, whereas dosages of 1 µL 100 g-1 compost were found to be efficacious for the volatiles. These optimized doses were assessed individually and in combined formulations with a hydrogel against oilseed rape (Brassica napus), sitka spruce (Picea sitchensis), maize (Zea mays) and strawberry (Fragaria annanassa). Both volatile compounds were highly effective biostimulants and were found to increase in biostimulatory efficiency when combined with M. brunneum conidia. Hydrogels were not found to interact with the growth process and may offer avenues for novel formulation technologies. This study demonstrates that Metarhizium-derived volatile organic compounds are actively involved in plant growth promotion and have potential for use in novel formulations to increase the growth of a wide range of commercially relevant crops.

Stress-Mediated Responses of Aedes aegypti (Diptera: Culicidae) Larvae When Exposed to Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Toxorhynchites brevipalpis (Diptera: Culicidae).

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds' population at risk from such diseases. Development of combined predator-parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite-predator treatments may be related to additive stress factors induced within the target host larvae.

Antibiotic Disk Elution Method to Determine the Colistin Susceptibility against Enterobacterales and Non-fermenter Bacteria.

Colistin minimum inhibitory concentration among Enterobacterales and Non-fermenters was determined using the new susceptibility method, Colistin Broth Disk Elution Method (CBDE), and its sensitivity and specificity. This descriptive cross-sectional study was conducted at Pakistan Railway Hospital, Rawalpindi from October 2020 to August 2021. Gram-negative bacteria were isolated and identified using Gram Stain and standard biochemical profile. Colistin Susceptibility was determined using CBDE and reference methods and then sensitivity and specificity of CBDE with standard reference methods. Essential and Categorical agreements were calculated. A total of 140 Gram-negative isolates were recovered from different specimens. The sensitivity and specificity of CBDE among Enterobacterales were 90.90% and 92.07% and for Pseudomonas aeruginosa 100% and 83.3% and for Acinetobacter baumannii 30% and 50% respectively. CBDE is simple, reliable, and cost effective to determine the colistin susceptibility among Enterobacterales and Pseudomonas aeruginosa while for Acinetobacter baumannii, this procedure is not useful. Key Words: Colistin susceptibility testing, CBDE, Enterobacterales, Non-fermenters.

Antimicrobial Volatiles of the Insect Pathogen Metarhizium brunneum.

Fungal volatile organic compounds (VOCs) represent promising candidates for biopesticide fumigants to control crop pests and pathogens. Herein, VOCs produced using three strains of the entomopathogenic fungus Metarhizium brunneum were identified via GC-MS and screened for antimicrobial activity. The VOC profiles varied with fungal strain, development state (mycelium, spores) and culture conditions. Selected VOCs were screened against a range of rhizosphere and non-rhizosphere microbes, including three Gram-negative bacteria (Escherichia coli, Pantoea agglomerans, Pseudomonas aeruginosa), five Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, B. megaterium, B. thuringiensis), two yeasts (Candida albicans, Candida glabrata) and three plant pathogenic fungi (Pythium ultimum, Botrytis cinerea, Fusarium graminearum). Microbes differed in their sensitivity to the test compounds, with 1-octen-3-ol and isovaleric acid showing broad-spectrum antimicrobial activity. Yeasts and bacteria were inhibited by the same VOCs. Cryo-SEM showed that both yeasts and bacteria underwent some form of "autolysis", where all components of the cell, including the cell wall, disintegrated with little evidence of their presence in the clear, inhibition zone. The oomycete (P. ultimum) and ascomycete fungi (F. graminearum, B. cinerea) were sensitive to a wider range of VOCs than the bacteria, suggesting that eukaryotic microbes are the main competitors to M. brunneum in the rhizosphere. The ability to alter the VOC profile in response to nutritional cues may assist M. brunneum to survive among the roots of a wide range of plant species. Our VOC studies provided new insights as to how M. brunneum may protect plants from pathogenic microbes and correspondingly promote healthy growth.

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

Evaluation of Steam-Ultrasound Decontamination on Naturally Contaminated Broilers through the Analysis of Campylobacter, Total Viable Count, and Enterobacteriaceae.

ABSTRACT: This study aimed to evaluate the decontamination effects of steam-ultrasound application, through specially designed nozzles installed inside a constructed machine, with a capacity of 10,500 birds per h on naturally contaminated broilers. Using three different skin-sampling areas-back, breast, and neck skin-microbial analysis of Campylobacter, Enterobacteriaceae, and total viable count (TVC) was performed before and after steam-ultrasound treatment. In total, 648 skin samples were analyzed for Campylobacter, and 216 samples were analyzed for Enterobacteriaceae and TVC. Results showed Campylobacter reductions (P < 0.001) of 0.8, 1.1, and 0.7 log, analyzed from back, breast, and the neck skin samples, respectively. Furthermore, reductions of Enterobacteriaceae (P < 0.001) by 1.6, 1.9, and 1.1 log and reductions of TVC (P < 0.001) by 2.0, 2.4, and 1.3 log were found on back, breasts, and neck, respectively. Campylobacter levels were evaluated after 8 days of refrigeration at 4°C in control and steam-ultrasound-treated broilers to determine contamination stability in a small 12-sample trial. The results showed no changes in reductions during refrigeration, indicating that reduced Campylobacter numbers remained stable in treated broilers. This study showed significant bacterial reduction was achieved in three different broiler surface areas at a slaughter speed of 10,500 birds per h at temperatures more than 80°C. The rapid treatment of less than 1.5-s exposure time inside the chamber makes this technology potentially suitable for modern and fast poultry processing lines.

Metarhizium anisopliae blastospores are highly virulent to adult Aedes aegypti, an important arbovirus vector.

BACKGROUND: The use of entomopathogenic fungi (EPF) for the control of adult mosquitoes is a promising alternative to synthetic insecticides. Previous studies have only evaluated conidiospores against adult mosquitoes. However, blastospores, which are highly virulent against mosquito larvae and pupae, could also be effective against adults. METHODS: Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores and conidia were first tested against adult Aedes aegypti by spraying insects with spore suspensions. Blastospores were then tested using an indirect contact bioassay, exposing mosquitoes to fungus-impregnated cloths. Virulence when using blastospores suspended in 20% sunflower oil was also investigated. RESULTS: Female mosquitoes sprayed with blastospores or conidia at a concentration of 108 propagules ml-1 were highly susceptible to both types of spores, resulting in 100% mortality within 7 days. However, significant differences in virulence of the isolates and propagules became apparent at 107 spores ml-1, with ESALQ 818 blastospores being more virulent than LEF 2000 blastospores. ESALQ 818 blastospores were highly virulent when mosquitoes were exposed to black cotton cloths impregnated with blastospores shortly after preparing the suspensions, but virulence declined rapidly 12 h post-application. The addition of vegetable oil to blastospores helped maintain virulence for up to 48 h. CONCLUSION: The results showed that blastospores were more virulent to adult female Ae. aegypti than conidia when sprayed onto the insects or applied to black cloths. Vegetable oil helped maintain blastospore virulence. The results show that blastospores have potential for use in integrated vector management, although new formulations and drying techniques need to be investigated.

Nanopore sequencing and de novo assembly of a misidentified Camelpox vaccine reveals putative epigenetic modifications and alternate protein signal peptides.

DNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.