Toll‐deficient Drosophila is susceptible to Pythium insidiosum infection

There is a paucity of animal models of pythiosis, a life‐threatening disease of humans and animals, the immunopathogenesis of which is poorly understood. A pythiosis model was developed by injecting Toll (Tl)‐deficient Drosophila melanogaster flies with Pythium insidiosum zoospores. The infected Tl mutant flies had significantly lower survival rates (73.7%) than did control flies. This study reveals the important role of Tl pathway activation in fly immune response to pythiosis.     

Intestinal canine pythiosis in Venezuela confirmed by serological and sequencing analysis

An 11-months-old mixed Terrier male originally from Venezuela, was referred to a Veterinary Hospital with signs of depression, anorexia, vomiting and diarrhea. The illness had begun 1 month earlier. Despite antibiotic chemotherapy and vitamins, the disease progressed. Radiological exams showed involvement of the small intestine. Histopathological studies of tissue samples taken during surgical intervention revealed eosinophilic areas in the center of which, abundant eosinophils, histiocytes and giant cells were observed. Silver stained cross-sections of the small intestine showed slender sparsely septate hyphae within the necrotic areas. Attempts to isolate the etiologic agent in pure culture were fruitless. The dog died without a definitive diagnosis. Fixed tissue samples of the small intestine were later investigated using specific fluorescent antibodies for pythiosis and molecular tools. These exams indicated that the hyphae in the infected tissues belong to the straminipilan pathogen Pythium insidiosum. This is the first confirmed case of dog pythiosis in Venezuela.     

Identification of <Emphasis Type="Italic">Pythium insidiosum</Emphasis> by Nested PCR in Cutaneous Lesions of Brazilian Horses and Rabbits

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.     

Human Pythiosis: Emergence of Fungal-Like Organism

Pythiosis is an emerging infectious disease caused by the aquatic oomycete Pythium insidiosum, a fungal-like organism. It is believed that P. insidiosum’s zoospores, its infected form, play major role in pathogenesis. Vascular and ocular infections are the most common clinical manifestation in humans. It is difficult to establish the diagnosis given its relatively rarity and difficulty to distinguish P. insidiosum from other molds. Delay in diagnosis and treatment has been associated with poor outcomes. High index of suspicion is the key, particularly in thalassemia patients with arterial insufficiency and patients with fungal keratitis/endophthalmitis without improvement on antifungal therapy. Tissue culture and zoospore induction remain gold standard for diagnosis; however, DNA-based method should be performed simultaneously. The combination of radical surgery, antifungal agents, and immunotherapy has been recommended. It was previously believed that surgery with negative surgical margins was the essential to survive in vascular pythiosis; however, it was recently found that patients could have residual disease despite documented negative surgical margins as infected clot may be dislodged to proximal arterial sites prior to surgery. Serum β-d-glucan (BG) has been used to monitor disease response after treatment initiation in vascular pythiosis. A significant decrease in BG levels within 2 weeks after surgery is indicative of the absence of residual infection. Unfortunately, monitoring tools for ocular pythiosis are not yet available. Itraconazole plus terbinafine have generally been used in P. insidiosum-infected patients; however, antibacterial agents, including azithromycin and linezolid, have also been used with favorable outcomes in ocular disease. Recently, azithromycin or clarithromycin plus doxycyclin were used in two relapsed vascular pythiosis patients with good outcomes.

     

Deploying strain specific hypersensitive resistance to diminish temporal virus spread

Spread of necrotic and non‐necrotic strains of Bean yellow mosaic virus (BYMV) was compared when aphid vectors moved both types from external or internal virus sources to plots of Lupinus spp. (lupin). Regardless of whether virus sources were internal or external, removed or left in place, and spread was within plots with homologous sources or across buffers to plots containing the opposite type of virus source, non‐necrotic BYMV always spread faster than necrotic BYMV in plots of L. angustifolius (narrow‐leafed lupin). When necrotic BYMV spread from external sources into plots sown with two L. angustifolius genotypes differing in their necrosis responses to different BYMV strain groups and one genotype of L. luteus (yellow lupin) giving only non‐necrotic responses, differing symptom reactions in the two L. angustifolius genotypes revealed presence of two distinct necrotic BYMV strain groups and overall virus spread was greater in this species than in L. luteus. Spread of non‐necrotic BYMV in L. angustifolius was always polycyclic in nature. However, when it came initially from external sources, spread of necrotic BYMV was largely monocyclic. This work demonstrates how temporal virus spread can be diminished when hypersensitive (necrotic) resistance is deployed and the limitations associated with employing hypersensitivity that is strain specific.     

Biological properties of necrotic and non-necrotic strains of bean yellow mosaic virus in cool season grain legumes

A collection of 51 bean yellow mosaic virus (BYMV) isolates was transmitted from infected Trifolium subterraneum (subterranean clover) to Lupinus angustifolius (narrow‐leafed lupin) by Myzus persicae (green peach aphid). Depending on isolate and L. angiistifolius genotype used, two distinct responses developed in L. angustifolius plants, either systemic necrosis and plant death or non‐necrotic reactions of varying severity. Ten isolates caused necrosis and plant death in cv. Danja. However, when nine of these were inoculated to breeding line 90L423‐07‐13, seven induced non‐necrotic reactions, while two caused necrosis and plant death. Thirty seven isolates always produced non‐necrotic reactions regardless of genotype of L. angustifolius inoculated. Non‐necrotic and necrotic isolates originally came both from lupins and other species, and the non‐necrotic isolates were no less efficiently transmitted by M. persicae than the necrotic ones. When one isolate of each type was inoculated together to T. subterraneum and nine months later this culture was used as an acquisition source for aphid transmission to L. angustifolius, only the necrotic type was detected. Previous infection of L. angustifolius plants with a non necrolic isolate prevented subsequent infection by a necrotic one. All necrotic and non‐necrotic isolates reacted with BYMV antiserum in ELISA but only two cross‐reacted with antiserum to clover yellow vein virus (CYVV). When selected necrotic and non‐necrotic isolates were inoculated to differential hosts, all behaved like BYMV and not CYVV. When three isolates of each type were transmitted to 11 other cool season grain legume species, except in Cicer arietinum (chickpea), there were no necrotic reactions, but symptom severity varied with the isolate and species inoculated. The two isolates that caused necrosis in C. arietinum did not do so in L. angustifolius. The six isolates from Vicia faba (faba bean) all caused non‐necrotic reactions in L. angustifolius cv. Danja and 90L423‐07‐13. These and two necrotic isolates readily infected five genotypes of V. faba always causing severe symptoms. However, three non‐necrotic isolates from L. angustifolius and a further necrotic isolate were poorly infectious on V. faba in which they generally induced mild symptoms. These results show that at least three strain groups of BYMV can be distinguished by their reactions in different L. angustifolius genotypes, one causing necrosis and death in cv. Danja and 90L423‐07‐13, one causing necrosis and death in Danja but not 90L423‐07‐13, and one causing non‐nccrotic reactions in both. These strain groups could not be distinguished when representative necrotic and non‐necrotic isolates were inoculated to other grain legume species. However, inoculation to V. faba distinguished two other BYMV strain groupings differing in severity of symptoms and ability to infect this species.     

Evaluation of two vaccines for the treatment of pythiosis insidiosi in horses

Two vaccines to treat pythiosis insidiosi in horses were evaluated in 71 Costa Rican horses between 1982 to 1988. One vaccine used a cell-mass (CMV) as antigen and the other a soluble concentrated antigen (SCAV). Both vaccines cured horses infected with Pythium insidiosum (p value 14%). The age of lesions prior to vaccination was important in the response of the horses to immunotherapy. All horses with lesions 0.5 months or less in duration were cured regardless of the vaccine used. Horses with lesions two or more months old did not respond to either vaccine. The age of the horses did not have any influence on their response to the vaccinations. The CMV produced a prominent inflammatory reaction at the side of injection, while the SCAV gave a low inflammatory reaction. In addition, the CMV lost its effectiveness two to three weeks after its preparation. By contrast, the SCAV maintained its ability to cure horses even after 18 months. Immunotherapy using SCAV can thus be used as the vaccine of choice in early cases of equine cutaneous pythiosis insidiosi.Private practice     

Immunodiffusion test for diagnosing human pythiosis

An immunodiffusion test was developed for diagnosing subcutaneous and systemic pythiosis in humans. When culture filtrate antigen (CFA) from P. insidiosum was reacted against patient and rabbit antisera, 1–5 precipitin bands occured both in patient and rabbit antisera, and a line of identity also occured between patient and rabbit sera. When control P. insidiosum CFA was reacted with 30 apparently normal persons, 20 Thalassemia patients, 2 candidosis and 5 aspergillosis patients, no precipitin bands were found. P. insidiosum CFA also tested with rabbit antibodies to B. dermatitidis, C. immitis, H. capsulatum, P. brasiliensis, C. albicans, M. furfur and A. fumigatus revealed no cross reactions. This test is practical, sensitive and specific.     

E‐ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy

Pythiosis is a life‐threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2‐like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto‐adenosine deaminase (E‐ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E‐ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E‐ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E‐ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy. Copyright © 2012 John Wiley & Sons, Ltd.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined.Objective. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL.Methods. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis.Results. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen.Conclusion. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.     

Interaction between 2,4-Dichlorophenoxyacetic Acid and Vitamin K in Datura stramonium

Plants of the cross Datura stramonium L. var. stramonium×Datura stramonium L. var. inermis (Juss. ex Jacq.) Timm. were sprayed either with a water solution of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D) or with a combination of 2,4-D (sodium salt) and the vitamin K compound menadione sodium bisulfite in water solution. The plants were found to differ in one of their responses to the treatments, namely the development of necrotic spots on the leaf blades. Many necroses appeared on the plants treated with 2,4-D plus menadione sodium bisulfite while only a few necroses or none at all developed on the plants treated with 2,4-D alone. Furthermore, 2,4-D in combination with menadione sodium bisulfite induced much larger necrotic spots than did 2,4-D by itself. Spraying with menadione sodium bisulfite in water solution did not produce any observable effects upon Datura plants. The great increase in necrotic lesion formation was probably due to an interaction between 2,4-D and menadione sodium bisulfite (or a metabolite of this compound) within leaf cells.     

Necrotic Lesions as Unusual Symptoms of Ring Rot in the Potato Leaves

Test-tube plants and suspension cell cultures of two cultivars of the potato (Solanum tuberosum L.) differing in their resistance to ring rot caused by Clavibacter michiganensis subsp. sepedonicus and six strains of this bacterium were used to test the relationship between the virulence, the leaf ability to adsorb bacteria, and the symptoms of the disease. In addition to chlorosis and drying, heavy inoculation with virulent strains caused unusual symptoms, such as leaf necrotic lesions. In the resistant cultivar, the necrotic lesions were predominantly local, whereas in the susceptible cultivar, they expanded. Unlike the susceptible cultivar, suspension cells of the resistant cultivar weakly adhered bacteria of the tested strains. Bacteria entered the plants through the leaf stomata. The sorption and penetration were much more pronounced in the susceptible cultivar. It was concluded that strain virulence varies depending on the conditions of inoculation, and uncharacteristic symptoms (necrotic lesions) arise. The local necrotic lesions are considered a hypersensitive response, and exopolysaccharides of the pathogen as the factors of virulence.     

Gastric pythiosis in a dog

BackgroundPythiosis is caused by the agent Pythium insidiosum, an aquatic oomycete of the kingdom Stramenopila.AimsTo describe the symptoms, pathological changes and diagnosis methods of gastric pythiosis in dogs.MethodsA three-year-old female German shepherd, with access to wetlands, was attended due to vomiting and recurrent diarrhea of 30 days of duration. A palpable mass in the abdomen filling the left epigastric region was identified in the clinical examination. Simple and contrasted radiological examination and ultrasound of abdominal cavity were performed. The animal was referred for exploratory laparotomy for the removal of the mass. The extent of the mass prevented from the excision and the animal was euthanized. Samples of the tumor mass were collected and sent for morphological study and immunohistochemistry.ResultsThe changes observed in imaging studies were consistent with gastric pythiosis. In cytology and histopathology, non-septate hyphae were identified, and in immunohistochemistry a strong positivity of anti-Pythium antibodies was observed, confirming the diagnosis of pythiosis.ConclusionsPythiosis in dogs is diagnosed late and tends to evolve in the animal's death. The definitive diagnosis is by cytology, histology and immunohistochemistry.     

Changes in endogenous cytokinin profiles in micropropagated <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> in relation to shoot-tip necrosis and cytokinin treatments

Changes in cytokinin (CK) profiles and their physiological implications in micropropagated Harpagophytum procumbens [(Burch.) DC. ex Meisn.] tissues in relation to shoot-tip necrosis (STN) and CK treatments were studied. Total CK content was quantified in benzyladenine (BA)-treated necrotic and normal plantlets and in plantlets treated with the CKs BA, meta-topolin (mT) and meta-topolin riboside (mTR) with and without the auxin indole-3-acetic acid (IAA). Generally necrotic shoots yielded more total CK compared to normal shoots. Cytokinin accumulation was higher at the basal section (basal > middle > top). Further analysis of the CKs based on structural and functional forms revealed excessive accumulation of 9-glucosides (deactivation products—toxic metabolites) and limited amounts of O-glucosides (storage forms—re-utilizable) in necrotic and BA-treated shoots compared to normal and topolin-treated cultures. The addition of IAA enhanced the formation of 9-glucosides in BA-treated cultures but reduced it in topolin-treated cultures. The symptom of STN could therefore be attributed to conversion of active cytokinins to other forms such as 9-glucosides which are neither active nor reversibly sequestrated to active forms. Literature shows that metabolites like 9-glucosides of BA have a detrimental effect in plant tissue culture.     

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and β-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Inheritance of hypersensitive resistance to Bean yellow mosaic virus in narrow-leafed lupin (Lupinus angustifolius)

In narrow‐leafed lupin (Lupinus angustifolius), segregation for the necrotic (systemic hypersensitive) response to infection with a necrotic strain of Bean yellow mosaic virus (BYMV‐N) was studied in progeny plants from six crosses. The parents were two cultivars that always developed necrosis when infected (Danja and Merrit) and two genotypes that always responded without necrosis (90L423‐07‐13 and P26697). In the four possible combinations of crosses between the different necrotic and non‐necrotically reacting genotypes, segregation for the necrotic response in F₂ progeny plants always fitted a 3:1 ratio (necrotic: non‐necrotic). All F₂ progeny plants from the cross between the two non‐cultivar genotypes became infected without necrosis while 99% of the F₂ from the cross between the two cultivars developed necrosis. These results indicate that the systemic necrotic response to infection with BYMV‐N is probably controlled by a single dominant hypersensitivity gene for which we propose the name Nbm‐1. However, its expression seemed influenced by independently segregating modifier genes in the genetic background since necrosis developed at widely different rates within affected F₂ progeny plants resulting in staggered killing.     

Complex Interaction of Deferasirox and Pythium insidiosum: Iron-Dependent Attenuation of Growth In Vitro and Immunotherapy-Like Enhancement of Immune Responses In Vivo

Pythium insidiosum iron acquisition mechanisms are unknown. We previously showed that the iron chelator deferasirox had weak activity in vitro and in rabbits with experimental pythiosis. Here we show that deferasirox causes damage to P. insidiosum hyphae in vitro, but that activity is diminished in the presence of exogenous iron. The tissue activity of the proinflammatory enzyme adenosine deaminase and the histological pattern observed in pythiosis lesions of rabbits treated with deferasirox were similar to the ones in animals treated with immunotherapy.     

Canine Gastrointestinal Pythiosis Treatment by Combined Antifungal and Immunotherapy and Review of Published Studies

Pythium insidiosum is an oomycete, a fungal like microorganism, which infects mammals, causing pythiosis in animals and humans, especially in tropical and subtropical regions around the world. The treatment for this infection is very difficult, and therapeutic options commonly comprise surgery, immunotherapy and antimicrobial drugs. The present report describes the clinical healing of a dog with gastrointestinal pythiosis by treatment with a combination of antifungals and immunotherapy, as well as reviews the cases reported in the literature that used some type of therapy for canine pythiosis. A 2.5-year-old male beagle initially showed sporadic vomiting episodes, and this symptom became more frequent 5 months after the onset of clinical signs. Celiotomy procedure found thickness of the stomach wall extending to the pylorus and duodenum. A biopsy was performed, and the diagnosis of pythiosis was made by mycological, histopathological analyses and molecular identification. Therapy was based on an association of terbinafine plus itraconazole during 12 months and immunotherapy for 2.5 months. The healing of the dog reported here allows us to propose the use of immunotherapy associated with antifungal therapy to treat canine gastrointestinal pythiosis. However, additional studies should be performed on a larger number of patients to establish a standard treatment protocol for canine pythiosis.     

Outdoor infrared imaging for spatial and temporal thermography: A case study of necrotic versus healthy leaf areas on woody plants

The temperature of plants can be measured using infrared (IR) thermography. Despite the extensive use of IR imaging indoors, outdoor IR imaging is uncommon. We used IR imaging to compare leaf temperatures between necrotic spots and healthy areas of oriental cherry (Prunus serrulata var. spontanea), Japanese cornel (Cornus officinalis) and sawtooth oak (Quercus acutissima) in the field. There was a significant difference in the mean leaf temperatures between the necrotic spots (26.4°C) and healthy areas (25.6°C) of oriental cherry (p = .01). The mean temperatures in the necrotic spots of Japanese cornel and sawtooth oak leaves were 22.3°C and 29.6°C, respectively, which were not significantly different from the mean temperatures of the healthy areas. A consecutive, 2‐day temporal leaf analysis in October 2018 revealed that the temperatures in the necrotic spots were generally higher than those in the healthy areas of the three species. The temperature difference between the spots and healthy areas (up to 1.4°C) was more pronounced at 13:00 hr in all three species on both days. These results reveal differences in the spatial and temporal thermal state across the necrotic spotted leaves. There is potential for use of outdoor IR imaging to visualize the response of trees to pathogen infection and abiotic stress.