Antifungal Prospect of Bacillus cereus Postbiotic on Crustacean Pathogen, Lagenidium thermophilum.

Pathogenic marine fungi, Lagenidium thermophilum is known causative agent in the crustacean industry. Current disinfection practice in hatchery has risks and negative impacts which prompts suitable substitute to synthetic antifungal agents. Thus, this study was conducted to evaluate the antifungal potential of postbiotic from four potential probiotics towards marine oomycetes, L. thermophilum IPMB 1401. The screening test showed that the Lactobacillus plantarum GS12 and Bacillus cereus GS15 postbiotics were positive for antifungal activity on L. thermophilum IPMB 1401. These two bacterial extracts have minimum inhibitory concentration （MIC） at 50%. The toxicity assay on MIC level of the postbiotic revealed that the cumulative mortality of brine shrimp nauplii exposed to B. cereus postbiotic was significantly lower compared to L. plantarum GS12 postbiotic and formalin. This indicates a high potential of B. cereus GS15 as a prospect for alternative control method for fungal infections in the crustacean culture industry.

Severe meningoencephalitis secondary to calvarial invasion of Lagenidium giganteum forma caninum in a dog.

Background: The oomycete Lagenidiumgiganteum forma caninum is an uncommon cause of severe dermal and subcutaneous infections in dogs with possible vascular invasion and other fatal sequelae. Infection within the central nervous system of affected dogs has not been previously reported. Case Description: A 6-year-old spayed female mixed-breed dog was evaluated at a referral institution with a 2-month history of suspected fungal infection in the region of the right mandibular lymph node that was refractory to surgical resection and empiric medical therapy. Physical examination identified a 6-cm fluctuant subcutaneous mass caudoventral to the ramus of the right mandible and a second firm mass in the region of the right caudal maxilla. Lesional punch biopsies were submitted for fungal culture and polymerase chain reaction (PCR), which subsequently identified L. giganteum forma caninum infection. Initial treatment consisted of anti-inflammatory doses of prednisone and hyperbaric oxygen therapy. Four weeks following initial evaluation, the patient was presented with progressive neurological signs consistent with a forebrain lesion. Magnetic resonance imaging revealed soft-tissue, contrast-enhancing lesions ventral to the calvarium adjacent to the site of original surgical resection and throughout the brain. Humane euthanasia was elected, and postmortem examination was consistent with the extension of local disease from the right masseter muscle into the right ventral calvarium. Postmortem DNA sequencing confirmed the identity of the organism as L. giganteum forma caninum. Conclusion: This is the first reported case of intracranial lagenidiosis in the dog. PCR distinguished this species from other Lagenidium species and from oomycetes of other genera, such as Pythiuminsidiosum and Paralagenidium karlingii. Regional extension of cutaneous lagenidiosis should therefore be considered in cases with concurrent or spontaneous neurologic disease.

Phylogenetic and physiological traits of oomycetes originally identified as Lagenidium giganteum from fly and mosquito larvae.

We studied phylogenetic and taxonomic features of isolates identified as Lagenidium giganteum recovered from six different species of mosquito larvae. The isolates grew vigorously at 25 C, moderately at 30 C, and not at all at 37 C and developed submerged, white colonies with few short, hyaline, aerial hyphae. Cultures displayed phenotypic plasticity, with broad, hyaline hyphae strongly constricted at septa that developed oval, spherical, or amorphous segments. These developed into sporangia producing one or two exit tubes, from which evanescent gelatinous vesicles containing zoospores developed. Three isolates developed oogonia consistent with features previously described for L. giganteum. Phylogenetic analysis of nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS = ITS) and cytochrome c oxidase subunit II (COXII) sequences of L. giganteum consistently grouped into eight clusters. Four of the investigated isolates grouped with sequences of an unnamed Lagenidium species infecting nematodes. Based on phenotypic and phylogenetic data, we describe the latter isolates as L. juracyae, sp. nov. In addition, we also investigated a species of Paralagenidium from a dog with lagenidiosis and describe it as new, Paralagenidium ajellopsis, sp. nov.

Nangallenes A and B, halogenated nonterpenoid C15-acetogenins from the Bornean red alga Laurencia nangii.

Two new halogenated nonterpenoids C15-acetogenins, nangallenes A-B (1-2), together with two known halogenated compounds itomanallene A (3) and 2,10-dibromo-3-chloro-α-chamigrene (4), were isolated and identified from the organic extract of the marine red alga Laurencia nangii Masuda collected from the coastal waters in Semporna, Borneo. Their structures were established by means of spectroscopic analysis including IR, high-resolution electrospray ionization mass spectrometry (HRESI-MS), and 1D and 2D NMR techniques. All these metabolites were submitted for the antifungal assay against four species of selected marine fungi. Compounds 1-4 showed potent activity against Haliphthoros sabahensis and Lagenidium thermophilum.

Leucoxenols A and B, two new phenolics from Bornean medicinal plant Syzygium leucoxylon.

The medicinal plant, Syzygium leucoxylon or commonly known as Obah found in North Borneo was considered as traditional medicine by local committee. Two new phenolics, leucoxenols A (1) and B (2) were isolated and identified as major secondary metabolites from the leaves of S. leucoxylon. Their chemical structures were elucidated based on spectroscopic data such as NMR and HRESIMS. Furthermore, these compounds were active against selected strains of fungi.

12-Epi-9-deacetoxyxenicin, new cytotoxic diterpenoid from a Bornean soft coral, Xenia sp.

One new compound, 12-epi-9-deacetoxyxenicin (1) along with a hydroperoxide product, 12-epi-9-deacetoxy-8-hydroperoxyxenicin (2) and two known sesquiterpenoids (3-4) were isolated from a population of Bornean soft coral Xenia sp. The structures of these secondary metabolites were elucidated based on their spectroscopic data. Compounds 1 and 2 showed cytotoxic activity against ATL cell line, S1T. In addition, compound 3 exhibited hyphal inhibition of Lagenidium thermophilum.

New cembrane-type diterpenoids from Bornean soft coral Nephthea sp. with antifungal activity against Lagenidium thermophilum.

Three new cembrane diterpenes, nephthecrassocolides A-B (1-2) and 6-acetoxy nephthenol acetate (3) along with three known compounds, 6-acetoxy-7,8-epoxy nephthenol acetate (4), epoxy nephthenol acetate (5) and nephthenol (6) were isolated from one population of Nephthea sp. Their structures were elucidated based on spectroscopic data analysis and the antifungal activities of compounds 1-6 were evaluated.

In vitro Inhibitory Effects of Two Bornean Medicinal Wild Gingers against Pathogenic Lagenidium thermophilum Infected Mud Crab Scylla tranquebarica.

　The antifungal activity of two Bornean medicinal wild gingers Plagiostachys megacarpa and Zingiber phillippsiae were examined against Lagenidium thermophilum. The most active extract was P. megacarpa at concentration of 320 µg/mL inhibiting both hyphal growth and zoospore production of L. thermophilum in 24 h. Toxicity tests were conducted using mud crab (Scylla tranquebarica) larva. Bath treatment of P. megacarpa at concentrations of 320 and 640 µg/mL for 24 h were highly effective against hyphae and zoospores of the strain and it is non-toxic to mud crab larva. Therefore, crude extracts P. megacarpa may be used as alternative treatment for marine Oomycete infection of mud crab.

Radioprotective potential of Lagenaria siceraria extract against radiation-induced gastrointestinal injury.

The cucurbits (prebiotics) were investigated as novel agents for radio-modification against gastrointestinal injury. The cell-cycle fractions and DNA damage were monitored in HCT-15 cells. A cucurbit extract was added to culture medium 2 h before irradiation (6 Gy) and was substituted by fresh medium at 4 h post-irradiation. The whole extract of the fruits of Lagenaria siceraria, Luffa cylindrica, or Cucurbita pepo extract enhanced G2 fractions (42%, 34%, and 37%, respectively) as compared with control (20%) and irradiated control (31%). With cucurbits, the comet tail length remained shorter (L. siceraria, 28 µm; L. cylindrica, 34.2 µm; C. pepo, 36.75 µm) than irradiated control (41.75 µm). For in vivo studies, L. siceraria extract (2 mg/kg body weight) was administered orally to mice at 2 h before and 4 and 24 h after whole-body irradiation (10 Gy). L. siceraria treatment restored the glutathione contents to 48.8 µmol/gm as compared with control (27.6 µmol/gm) and irradiated control (19.6 µmol/gm). Irradiation reduced the villi height from 379 to 350 µm and width from 54 to 27 µm. L. siceraria administration countered the radiation effects (length, 366 µm; width, 30 µm, respectively) and improved the villi morphology and tight junction integrity. This study reveals the therapeutic potential of cucurbits against radiation-induced gastrointestinal injury.

Glycoside hydrolases family 20 (GH20) represent putative virulence factors that are shared by animal pathogenic oomycetes, but are absent in phytopathogens.

BACKGROUND: Although interest in animal pathogenic oomycetes is increasing, the molecular basis mediating oomycete-animal relationships remains virtually unknown. Crinkler (CRN) genes, which have been traditionally associated with the cytotoxic activity displayed by plant pathogenic oomycetes, were recently detected in transcriptome sequences from the entomopathogenic oomycete Lagenidium giganteum, suggesting that these genes may represent virulence factors conserved in both animal and plant pathogenic oomycetes. In order to further characterize the L. giganteum pathogenome, an on-going genomic survey was mined to reveal novel putative virulence factors, including canonical oomycete effectors Crinkler 13 (CRN13) orthologs. These novel sequences provided a basis to initiate gene expression analyses and determine if the proposed L. giganteum virulence factors are differentially expressed in the presence of mosquito larvae (Aedes aegypti). RESULTS: Sequence analyses revealed that L. giganteum express CRN13 transcripts. The predicted proteins, like other L. giganteum CRNs, contained a conserved LYLA motif at the N terminal, but did not display signal peptides. In contrast, other potential virulence factors, such as Glycoside Hydrolases family 20 (hexosaminidase) and 37 (trehalase) proteins (GH20 and GH37), contained identifiable signal peptides. Genome mining demonstrated that GH20 genes are absent from phytopathogenic oomycete genomes, and that the L. giganteum GH20 sequence is the only reported peronosporalean GH20 gene. All other oomycete GH20 homologs were retrieved from animal pathogenic, saprolegnialean genomes. Furthermore, phylogenetic analyses demonstrated that saprolegnialean and peronosporalean GH20 protein sequences clustered in unrelated clades. The saprolegnialean GH20 sequences appeared as a strongly supported, monophyletic group nested within an arthropod-specific clade, suggesting that this gene was acquired via a lateral gene transfer event from an insect or crustacean genome. In contrast, the L. giganteum GH20 protein sequence appeared as a sister taxon to a plant-specific clade that included exochitinases with demonstrated insecticidal activities. Finally, gene expression analyses demonstrated that the L. giganteum GH20 gene expression level is significantly modulated in the presence of mosquito larvae. In agreement with the protein secretion predictions, CRN transcripts did not show any differential expression. CONCLUSIONS: These results identified GH20 enzymes, and not CRNs, as potential pathogenicity factors shared by all animal pathogenic oomycetes.

Molecular phylogeny and taxonomy of Lagenidium-like oomycetes pathogenic to mammals.

Over the past twenty years, infections caused by previously unrecognised oomycete pathogens with morphological and molecular similarities to known Lagenidium species have been observed with increasing frequency, primarily in dogs but also in cats and humans. Three of these pathogens were formally described as Lagenidium giganteum forma caninum, Lagenidium deciduum, and Paralagenidium karlingii in advance of published phylogenetic verification. Due to the complex nature of Lagenidium taxonomy alongside recent reports of mammalian pathogenic species, these taxa needed to be verified with due consideration of the available data for Lagenidium and its allied genera. This study does so through morphologic characterisation of the mammalian pathogenic species, and phylogenetic analyses. The six-gene phylogeny generally supports the most recent comprehensive classification of Lagenidium with a well-supported Lagenidium clade that includes the mammalian pathogens L. giganteum f. caninum and L. deciduum, and well-supported clades for which the names Myzocytiopsis and Salilagenidium can be applied. The genus Paralagenidium is phylogenetically unrelated to any of the main clades within the class Peronosporomycetes. Close relationships between pathogens of mammals and those of insects or nematodes were revealed. Further characterisation of Lagenidium-like taxa is needed to establish the risk of mammalian infection by pathogens of insects and nematodes.

Description of three novel Lagenidium (Oomycota) species causing infection in mammals / Descripción de tres nuevas especies del género Lagenidium (Oomycota) responsables de infecciones en mamíferos

Background. Recent molecular phylogenetic analysis of Lagenidium strains recovered from subcutaneous lesions in cats, dogs, and a human with lagenidiosis resolved into four clades; one of them was Lagenidium giganteum, but three others were novel. Aims. Due to the recent increase in L. giganteum infections from mammals, we studied 21 Lagenidium strains isolated from dogs and a human available in our collection. Methods. Molecular phylogenetic studies and phenotypic characteristics were used to characterize the strains. Results. We report the finding of three novel species, herein designated as Lagenidium ajelloi, sp. nov., Lagenidium albertoi sp. nov, and Lagenidium vilelae sp. nov. Their morphological and growth features are also presented. Conclusions. Our study revealed the presence of three novel Lagenidium species infecting mammals (AU)

Antecedentes. Estudios recientes, basados en análisis filogenéticos, han revelado la existencia de cuatro clados de Lagenidium aislados en lesiones subcutáneas de gatos, perros y de un ser humano con lagenidiosis. Uno de los aislamientos se identificó como Lagenidium giganteum, pero los otros tres se consideraron especies nuevas. Objetivos. Debido al incremento de las infecciones por L. giganteum en mamíferos, se han estudiado 21 aislamientos de Lagenidium procedentes de animales con lagenidiosis. Métodos. Los aislamientos se clasificaron fenotípicamente, además de llevar a cabo estudios de filogenia con ellos. Resultados. Se proponen tres nuevas especies de Lagenidium: Lagenidium ajelloi, sp. nov., Lagenidium albertoi, sp. nov., y Lagenidium vilelae, sp. nov. Se tratan también sus características morfológicas. Conclusiones. Nuestro estudio reveló la existencia de tres especies nuevas de Lagenidium responsables de infecciones en mamíferos (AU)

Description of three novel Lagenidium (Oomycota) species causing infection in mammals.

BACKGROUND: Recent molecular phylogenetic analysis of Lagenidium strains recovered from subcutaneous lesions in cats, dogs, and a human with lagenidiosis resolved into four clades; one of them was Lagenidium giganteum, but three others were novel. AIMS: Due to the recent increase in L. giganteum infections from mammals, we studied 21 Lagenidium strains isolated from dogs and a human available in our collection. METHODS: Molecular phylogenetic studies and phenotypic characteristics were used to characterize the strains. RESULTS: We report the finding of three novel species, herein designated as Lagenidium ajelloi, sp. nov., Lagenidium albertoi sp. nov, and Lagenidium vilelae sp. nov. Their morphological and growth features are also presented. CONCLUSIONS: Our study revealed the presence of three novel Lagenidium species infecting mammals.

A biochemical screening approach to putatively differentiate mammalian pathogenic Oomycota species in the clinical laboratory.

The report of four novel mammalian pathogenic species of the genus Lagenidium prompted us to study the use of biochemical assays to differentiate the Oomycota mammalian pathogens Pythium insidiosum and Lagenidium spp. We investigated the reaction of 23 Lagenidium and eight Pythium species in various biochemical assays. Because the morphological features of the Oomycota species are similar to those of species in the Entomophthoramycota and Mucormycota, five fungal species with coenocytic hyphae were also included. We found that mammalian and plant isolates of Pythium spp. all hydrolysed sucrose, but Lagenidium species and the fungal strains did not. In addition, both Pythium spp. and Lagenidium spp. were found to be maltose-positive, whereas fungal strains did not hydrolyse this sugar. The fungal species and thermo-sensitive Lagenidium giganteum and Lagenidium humanum were urease-negative, but the mammalian Lagenidium spp. and Pythium spp. hydrolysed urea within 24  h. These findings suggest these assays can be used for the presumptive differentiation of mammalian Oomycota species in the laboratory.

Lagenidium giganteum pathogenicity in mammals.

Infections of mammals by species in the phylum Oomycota taxonomically and molecularly similar to known Lagenidium giganteum strains have increased. During 2013-2014, we conducted a phylogenetic study of 21 mammalian Lagenidium isolates; we found that 11 cannot be differentiated from L. giganteum strains that the US Environmental Protection Agency approved for biological control of mosquitoes; these strains were later unregistered and are no longer available. L. giganteum strains pathogenic to mammals formed a strongly supported clade with the biological control isolates, and both types experimentally infected mosquito larvae. However, the strains from mammals grew well at 25°C and 37°C, whereas the biological control strains developed normally at 25°C but poorly at higher temperatures. The emergence of heat-tolerant strains of L. giganteum pathogenic to lower animals and humans is of environmental and public health concern.

Development and evaluation of an ELISA for the quantitation of anti-Lagenidium giganteum forma caninum antibodies in dogs.

BACKGROUND: Lagenidium giganteum forma caninum infection causes severe cutaneous and disseminated disease in dogs. Currently, diagnosis requires culture and rRNA gene sequencing. OBJECTIVE: To develop and evaluate an ELISA for quantitation of anti-L. giganteum f. caninum IgG in canine serum. ANIMALS: Sera were evaluated from 22 dogs infected with L. giganteum f. caninum, 12 dogs infected with Paralagenidium karlingii, 18 dogs infected with Pythium insidiosum, 26 dogs with nonoomycotic fungal infections or other cutaneous or systemic diseases, and 10 healthy dogs. METHODS: Antigen was prepared from a soluble mycelial extract of L. giganteum f. caninum. Optimal antigen and antibody concentrations were determined by checkerboard titration. Results were expressed as percent positivity (PP) relative to a strongly positive control serum. RESULTS: Medians and ranges for PP for each group were: L. giganteum f. caninum (73.9%, 27.9-108.9%), P. karlingii (55.0%, 21.0-90.6%), P. insidiosum (31.3%, 15.8-87.5%), nonoomycotic fungal infection or other cutaneous or systemic diseases (19.2%, 3.2-61.0%), and healthy dogs (9.9%, 7.6-24.6%). Using a PP cutoff value of 40%, sensitivity and specificity (with 95% CI) of the ELISA for detecting L. giganteum f. caninum infection in clinically affected dogs were 90.9% (72.2-97.5%) and 73.2% (60.4-83.0%), respectively. Specificity in dogs infected with P. karlingii was 41.7% (19.3-68.1%) and with P. insidiosum was 66.7% (43.8-83.7%). CONCLUSIONS AND CLINICAL IMPORTANCE: Quantitation of anti-L. giganteum f. caninum antibodies for detection of this infection in dogs has moderately high sensitivity but poor specificity, the latter because of substantial cross-reactivity with anti-P. karlingii and anti-P. insidiosum antibodies.

Transcriptome analysis of the entomopathogenic oomycete Lagenidium giganteum reveals putative virulence factors.

A combination of 454 pyrosequencing and Sanger sequencing was used to sample and characterize the transcriptome of the entomopathogenic oomycete Lagenidium giganteum. More than 50,000 high-throughput reads were annotated through homology searches. Several selected reads served as seeds for the amplification and sequencing of full-length transcripts. Phylogenetic analyses inferred from full-length cellulose synthase alignments revealed that L giganteum is nested within the peronosporalean galaxy and as such appears to have evolved from a phytopathogenic ancestor. In agreement with the phylogeny reconstructions, full-length L. giganteum oomycete effector orthologs, corresponding to the cellulose-binding elicitor lectin (CBEL), crinkler (CRN), and elicitin proteins, were characterized by domain organizations similar to those of pathogenicity factors of plant-pathogenic oomycetes. Importantly, the L. giganteum effectors provide a basis for detailing the roles of canonical CRN, CBEL, and elicitin proteins in the infectious process of an oomycete known principally as an animal pathogen. Finally, phylogenetic analyses and genome mining identified members of glycoside hydrolase family 5 subfamily 27 (GH5_27) as putative virulence factors active on the host insect cuticle, based in part on the fact that GH5_27 genes are shared by entomopathogenic oomycetes and fungi but are underrepresented in nonentomopathogenic genomes. The genomic resources gathered from the L. giganteum transcriptome analysis strongly suggest that filamentous entomopathogens (oomycetes and fungi) exhibit convergent evolution: they have evolved independently from plant-associated microbes, have retained genes indicative of plant associations, and may share similar cores of virulence factors, such as GH5_27 enzymes, that are absent from the genomes of their plant-pathogenic relatives.

Lagenidium sp. ocular infection mimicking ocular pythiosis.

This is a report of a Lagenidium sp. in a Thai patient who was diagnosed with severe keratitis that was unresponsive to antibacterial and antifungal drugs. Examination of a corneal biopsy specimen confirmed the presence of aseptate hyphae. The internal transcribed spacer DNA sequence of the strain isolated showed 97% identity with Lagenidium giganteum and other Lagenidium species.

Microorganism viability influences internal phase droplet size changes during storage in water-in-oil emulsions.

Water-in-oil emulsions provide an alternative for long-term stabilization of microorganisms. Maintaining physical stability of the emulsion and cell viability is critical for large-scale application. Water-in-oil (W/O) emulsions were prepared with the biolarvacide Lagenidium giganteum and the green alga Chlorella vulgaris. Physical stability was measured via light scattering measurements of the internal phase droplets and cell viability was measured by plating and enumerating colony forming units. Emulsions were demonstrated to stabilize L. giganteum and C. vulgaris for more than 4 months without refrigeration. Introducing nutrients into the internal phase of W/O emulsions without cells had no significant effect on changes in aqueous phase droplet size dynamics. Internal phase droplet size changes that occurred over time were greater in the presence of cells. Increases in droplet size were correlated with cell death indicating measurement of internal phase droplet size changes may be an approach for monitoring declines in cell viability during storage.