Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

Cryptococcus: isolamento ambiental e caracterização bioquímica / Cryptococcus: environmental isolation and biochemical characterization

O gênero Cryptococcus caracteriza-se por ser uma levedura responsável por infecção sistêmica, causada pelas espécies Cryptococcus neoformans e Cryptococcus gattii. O fungo é encontrado em substratos de origem animal e vegetal, e a infecção ocorre com a inalação de basidiósporos ou leveduras desidratadas infectantes presentes no ambiente. O presente trabalho teve por objetivo pesquisar a existência de microfocos de Cryptococcussp.em amostras ambientais da cidade de Araçatuba, São Paulo, com a finalidade de minimizar os riscos de contaminação do homem e dos animais, buscando o conhecimento da ecoepidemiologia do Cryptococcus. Foram colhidas 50 amostras oriundas de ocos e troncos de árvores (Cassiasp., Ficussp., Caesalpinea peltophorides) de 10 locais representativos do perímetro urbano, as quais foram encaminhadas ao Laboratório de Bacteriologia e Micologia da Faculdade de Medicina Veterinária de Araçatuba-Unesp, onde foram processadas e semeadas em placas de Petri contendo ágar semente de Níger e Sabouraud dextrose com clorafenicol e incubadas à temperatura de 30ºC, por um período não inferior a cinco dias. Posteriormente, foram submetidas às provas bioquímicas: produção de urease, termotolerância a 37ºC e quimiotipagem em ágar CGB (L-canavanina-glicina-azul de bromotimol). A análise dos resultados revelaram que 17 (34%) dos cultivos foram positivos para o gênero Cryptococcus, sendo nove (18%) para Cryptococcus gattiie oito (16%) para Cryptococcus neoformans. Outras leveduras correlacionadas, como Rhodotorula sp. e Candida sp., também foram isoladas. Conclui-se que os basidiósporos de Cryptococcusencontram-se dispersos na natureza, constituindo microfocos ambientais, não vinculados necessariamente a um único hospedeiro.

Cryptococcosis is an opportunistic fungal infection caused by Cryptococcusyeasts, especially C. neoformans and Cryptococcus gattii. The fungus is found in substrates of animal and vegetable origin, and infection occurs through inhalation and seedlings present in the environment. The present study aimed to investigate the existence of microfocus Cryptococcus sp. from the environmental samples of Araçatuba city, São Paulo, featuring new niches, by decoupling the direct relationship between fungus and host in order to minimize the risk of contamination of man and animals, understanding the ecoepidemiology of Cryptococcus. Fifty samples from hollows and tree trunks were harvested (Cassia sp., Ficus sp., Caesalpinea peltophorides) from ten representatives in the urban perimeter. The samples were immediately sent to the Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine Araçatuba - Unesp where they were processed and plated on Petri dishes containing agar seed Niger and Sabouraud dextrose agar with chloramphenicol, incubated at 30ºC for a period of no less than 5 days. Afterwards they were subimitted to biochemical tests: urease production, thermotolerance at 37°C and quimiotipagem in CGB agar (L- Canavanine-Glycine-Bromothymol blue). The results showed that 17 (34%) cultures were positive for Cryptococcus, 9 (18%) for Cryptococcus gattii and 8 (16%) for Cryptococcus neoformans. Other yeast correlated as Rhodotorula sp. and Candida sp. were isolated. We conclude that the infectious propagules of Cryptococcus are dispersed in nature and constitute an environmental microfocus, not necessarily being bound to a single host.

Ocurrencia natural de hongos entomophthorales de áfidos plaga de Medicago sativa L. en Argentina / Natural occurrence of entomophthoroid fungi of aphid pests on Medicago sativa L. in Argentina

Se encontraron cuatro especies de hongos Entomophthorales, Pandora neoaphidis, Zoophthora radicans, Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) y Neozygites fresenii (Neozygitales: Neozygitaceae) infectando a Aphis craccivora, Therioaphis trifolii, Acyrthosiphon pisum y a especies no identificadas pertenecientes al género Acyrthosiphon en cultivos de alfalfa (Medicago sativa L.), en la Argentina. Los muestreos fueron realizados en cinco sitios (Ceres, Rafaela, Sarmiento, Monte Vera y Bernardo de Irigoyen) de la provincia de Santa Fe. Zoophthora radicans fue el patógeno más importante registrado principalmente en Acyrthosiphon sp. Zoophthora radicans fue exitosamente aislado y mantenido en cultivos puros. Este estudio documenta por primera vez en la Argentina la presencia de hongos Entomophthorales infectando áfidos en alfalfa

Four species of entomophthoroid fungi, Pandora neoaphidis (Entomophthorales: Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae), Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) and Neozygites fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora, Therioaphis trifolii, and Acyrthosiphon pisum and unidentified species of Acyrthosiphon on lucerne in Argentina. Samples were collected from five sites (Ceres, Rafaela, Sarmiento, Monte Vera and Bernardo de Irigoyen) in the province of Santa Fe. In this study, Zoophthora radicans was the most important pathogen and was recorded mainly on Acyrthosiphon sp. Zoophthora radicans was successfully isolated and maintained in pure cultures. This study is the first report of entomophthoroid fungi infecting lucerne (Medicago sativa L.) aphids in Argentina

Susceptibility of Gyropsylla spegazziniana (Lizer & Trelles) (Hemiptera: Psyllidae) to Beauveria bassiana (Bals. ) Vuill / Suscetibilidade de Gyropsylla spegazziniana (Lizer & Trelles) (Hemiptera: Psyllidae) a Beauveria bassiana (Bals. ) Vuill

Gyropsylla spegazziniana is one of the most prominent pest insects of yerba mate culture in all production regions in Brazil, Argentina and Paraguay. Natural enemies have been recorded on G. spegazziniana nymphs and adults, including the Zoophthora radicans entomopathogenic fungus. Since there are no reports of Beauveria bassiana, the aim of this study was to register its pathogenicity with respect to this insect in a laboratory setting. Yerba mate branches were kept in glass flasks with water and we infested each leaf with 20 newly-emerged nymphs. We prepared three replicates per treatment. We sprayed conidia suspensions (1 × 109 conidia/mL) onto the branches, which we transferred to cages and kept in an acclimatized room (26 ± 1°C, 70 ± 10% of relative humidity (R.H.) with a photophase of 14 hours). We evaluated insect mortality daily, and after five days we observed a high level of pathogenicity due to the presence of the fungus, that could be visually observed on the cadaver. The total mortality varied from 25 to 70% (respectively for Unioeste 4 and CG 716) and the confirmed mortality was 30% for Unioeste 52, revealing the fungus' potential. However, more studies are necessary in order to evaluate the strains of this fungus, as well as other species.

Gyropsylla spegazziniana é uma das principais pragas da cultura de erva-mate em todas as regiُes produtoras do Brasil, da Argentina e do Paraguai. Inimigos naturais têm sido registrados sobre ninfas e adultos de G. spegazziniana, incluindo o fungo entomopatogênico Zoophthora radicans. Como nمo hل relatos sobre Beauveria bassiana, o objetivo deste estudo foi registrar sua patogenicidade sobre esse inseto em condiçُes de laboratَrio. Ramos de erva-mate foram infestados com insetos e mantidos em frascos de vidro com لgua. Cada um dos ramos tinha cerca de 20 ninfas recém-emergidas/folha, sendo preparadas 3 repetiçُes/tratamento. As suspensُes dos isolados do fungo (1 × 109 conيdios/mL) foram pulverizadas sobre os ramos, que foram transferidos para gaiolas e mantidos em sala climatizada (26 ± 1°C, 70 ± 10% de Umidade Relativa (U.R.) e 14 horas de fotofase). Diariamente, realizou-se a avaliaçمo da mortalidade do inseto, observando-se, apَs cinco dias da aplicaçمo, alto nيvel de patogenicidade devido à presença do fungo, constatada visualmente sobre os cadلveres. A mortalidade total variou entre 25 e 70% (respectivamente para Unioeste 4 e CG 716) e a mortalidade confirmada foi de 30% para o isolado Unioeste 52, revelando o potencial do fungo. No entanto, mais estudos sمo necessلrios para avaliar novos isolados do fungo, bem como outras espécies.

child with intestinal basidiobolomycosis

Fungal infections of the gastrointestinal tract are not common in children, especially in immunocompetent ones. In this case report we describe a child who was presented with abdominal pain and mass, bloody diarrhea and fever. He was treated for amebiasis, but due to treatment failure and deterioration of his condition, he underwent a laparatomy. Histologic examination of the excised bowel in the second look revealed Basidiobolomycosis, a fungus belonging to the order Entomophthorales. The signs, symptoms, treatment and diagnosis of the present case indicate that fungal infections must be considered not only in immunocompromised patients with abdominal pain and mass, but also in apparently immunocompetent ones

Intestinal obstruction due to Basidiobolus ranarum: An unusual case.

A case of intestinal obstruction caused by extensive soft tissue fungal infection of the perineum due to Basidiobolus ranarum is presented here. There was excellent response to antifungal treatment. A literature search revealed the case report of intestinal obstruction due to intrinsic mucosal involvement by the fungus, but extensive soft tissue involvement of the perineum resulting in extraneous obstruction to the rectum, has not been reported so far.

Meningoencephalitis caused by a zygomycete fungus (Basidiobolus) associated with septic shock in an immunocompetent patient: 1-year follow-up after treatment

Zygomycosis is an infection caused by opportunistic fungi of the Zygomycetes class, specifically those from the Mucorales and Entomophthorales orders. It is an uncommon disease, mainly restricted to immunocompromised patients. We report a case of a 73-year-old male patient with a history of fever (39°C) lasting for 1 day, accompanied by shivering, trembling, and intense asthenia. The patient was admitted to the intensive care unit with complex partial seizures, and submitted to orotracheal intubation and mechanical ventilation under sedation with midazolam. The electroencephalogram showed evidence of non-convulsive status epilepticus. There is no fast specific laboratory test that permits confirmation of invasive fungal disease. Unless the physician suspects this condition, the disease may progress rapidly while the patient is treated with broad-spectrum antibiotics. Differential diagnosis between fungal and bacterial infection is often difficult. The clinical presentation is sometimes atypical, and etiological investigation is not always successful. In the present case, the histopathological examination of the biopsy obtained from the right temporal lobe indicated the presence of irregular, round, thick-walled fungi forming papillae and elongated structures of irregular diameter, with no septa, indicative of zygomycete (Basidiobolus). Treatment with liposomal amphotericin B and fluconazole was initiated after diagnosis of meningoencephalitis by zygomycete, with a successful outcome.

Basidiobolomycosis: a case report

Basidiobolomycosis is a rare disease caused by the fungus Basidiobolus ranarum. Patients with Basidiobolus ranarum infection may present with subcutaneous, gastrointestinal, or systemic lesions. Recently, its etiologic role in gastrointestinal infections has been increasingly recognized. We report this case of gastrointestinal and retroperitoneal basidobolomycosis because of its rarity and also to emphasize the need to consider gastrointestinal and retroperitoneal basidiobolomycosis in the differential diagnosis of inflammatory bowel diseases, tuberculosis and malignancy

Primeiro Registro de Zoophthora radicans (Entomophthorales: Entomophthoraceae) em Adultos da Ampola-da-Erva-Mate, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae), no Brasil / First Record of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Adults of the Paraguay Tea Psyllid, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae), in Brazil

A primeira ocorrência de Zoophthora radicans (Entomophthorales: Entomophthoraceae) infectando adultos de Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae) em plantio comercial de erva-mate (Ilex paraguariensis), em Cascavel, PR, Brasil, é relatada. A prevalência do fungo foi elevada (90 por cento de mortalidade), sendo considerada uma epizootia natural.

We report the first occurrence of Zoophthora radicans infecting adults of Gyropsylla spegazziniana Lizer & Trelles in a commercial Paraguay tea plantation (Ilex paraguariensis), in Cascavel, PR, Brazil. The fungus prevalence was high (90 percent of mortality), considered a natural epizooty.

Basidiobolomycosis: a rare case report.

We report a rare case of basidiobolomycosis seen in an 11-year-old girl from North-Eastern part of India. She presented with complaints of bilateral nasal block and nasal discharge for seven-eight months. CT scan of sinuses revealed polypoidal mass in all the sinuses with extradural extension. The tissue biopsy examined histopathologically and microbiologically, revealed Basidiobolus ranarum.

Outbreaks of rhinofacial and rhinopharyngeal zygomycosis in sheep in Paraíba, northeastern Brazil

Two outbreaks of zigomycosis with rhinofacial and two other with rhinopharyngeal lesions involving fungi with filamentous coaenocytic hyphae characteristic of entomoph-thoramycetous fungi are reported in the state of Paraíba, northeastern Brazil. One outbreak of rhinofacial zygomycosis occurred during the rainy season affecting 5 sheep. Another outbreak of the clinical form affected one out of 40 sheep during the dry season. Common clinical signs of the rhinofacial infection were bilateral serosanguineous nasal discharge with swelling of nostrils, upper lip, and the skin of the face. At necropsy the nasal mucosa showed dark brownish ulcerated areas which extended from the mucocutaneous region to 10cm inside the nasal vestibule. The mucosa of the hard palate was also ulcerated. The cutting surface of nostrils and palate showed a brownish or red spongeous tissue of friable consistency. One outbreak of rhinopharyngitis took place on an irrigated coconut farm; 7 out of 60 adult sheep were affected. Another outbreak affected a sheep in a flock of 80 during the dry season. Clinical signs as noisy respiration and dyspnoea due to mechanical blockage of the nasal cavities, swelling of the nostrils, and serosanguineous nasal discharge were observed. Six out of 8 sheep in this group showed exophthalmia, keratitis and unilateral corneal ulceration of the eye. The sheep either died of their infection or were euthanized after a clinical course of 7-30 days. At necropsy there was a dense yellow exudate in the nasopharyngeal area affecting the ethmoidal region, turbinate bones, paranasal sinuses, hard and soft palates, orbital cavity, pharynges, regional muscles and lymph nodes. Histopathologically both forms of the disease showed multifocal granulomas with an eosinophilic necrotic reaction (Splendore-Hoeppli phenomenon) containing ribbon-type coenocytic hyphae with 7-30mm in diameter similar to hyphae of zygomycetous fungi, possibly Conidiobolus spp. Outbrea...

Dois surtos de lesões rinofaciais e dois de lesões rinofaríngeas causadas por fungos com hifas filamentosas, raramente septadas, semelhantes às dos fungos entomophthorales, são descritos no Estado da Paraíba. Um surto da forma rinofacial afetou 5 ovinos durante a estação chuvosa. Outro surto desta forma clínica afetou um ovino de um grupo de 40, durante a época seca. Os sinais clínicos da forma rinofacial foram de corrimento serosanguinolento bilateral, com aumento de volume das narinas, lábio superior, e pele da face. Na necropsia, a mucosa nasal apresentava áreas ulceradas de cor marrom escuro, que se estendiam desde a região muco-cutánea até 10cm dentro do vestíbulo nasal. A mucosa do palato duro estava, também, ulcerada. A superfície de corte das narinas e palato apresentava-se marrom ou avermelhada de aspecto esponjoso e friável. Um surto de rinofaringite micótica ocorreu em uma plantação de coco irrigado, afetando 7 ovinos de um rebanho de 60 ovinos adultos. Outro surto desta forma da enfermidade afetou um único animal de um rebanho de 80, durante a época seca. Os animais apresentavam dispnéia, com respiração ruidosa devida ao bloqueio parcial das narinas, e corrimento nasal serosanguinolento. Seis dos oito animais afetados por esta forma da enfermidade apresentaram, em um olho, exoftalmia, ceratite e ulceração da córnea. Todos os ovinos morreram ou foram sacrificados após um curso clínico de 7-30 dias. Na necropsia exsudato amarelo consistente foi observado na região etmoidal, ossos turbinados, seios paranasais, palatos duro e mole, órbita, faringe, e músculos e linfonodos regionais. No estudo histológico, ambas as formas da doença mostraram granulomas multifocais com áreas necróticas eosinofílicas (material de Splendore-Hoeppli) contendo hifas com 7-30mm de diâmetro, raramente septadas, semelhantes às dos fungos entomophthorales, possivelmente Conidiobolus spp. Surtos de ambas as formas de rinite micótica são freqüentes na região ...

Rhinoentomophthoromycosis.

A sixty year old patient presented with a slowly progressive swelling of the nose, of one year duration, suggesting a clinical diagnosis of subcutaneous zygomycosis. On investigation, the tissue fungal culture grew Conidiobolus coronatus, confirming the diagnosis as rhinoentomophthoromycosis. He was treated with a combination of oral fluconazole and oral potassium iodide for a total period of 5 months. His symptoms subsided completely. Serial CT scanning of paranasal sinuses showed the gradual resolution of the swelling, in response to the treatment. Early detection of the disease and combination therapy gave rapid and good results. This is the first case of its kind to be reported from Kerala, the southern state of India.

Hongos patógenos de insectos en Argentina (Zygomycetes: Entomophthorales) / Pathogenic fungi of insects from Argentina (Zygomycetes: Entomophthorales)

Three species of Entomophthorales entomopathogenic fungi (Zygomycotina: Zygomycetes) have been identified from insects in agricultural crops (Buenos Aires Province, Argentina): Zoophthora radicans Batko (Brefeld); Entomophthora planchoniana Cornu and Pandora gammae (Weiser) Humber. Fungal structure measurements are reported.

Se informa de tres especies de hongos Entomophthorales en insectos encontrados en cultivos agrícolas en la Provincia de Buenos Aires, Argentina: Zoophthora radicans Batko (Brefeld); Entomophthora planchoniana Cornu y Pandora gammae (Weiser) Humber. Se incluyen las medidas de las estructuras fúngicas.

Case report of subcutaneous entomophthoromycosis with retroperitoneal invasion

Os autores descrevem um caso de entomoftoromicose em paciente previamente saudável, que apresentou abscesso em nádega direita, evoluindo, após drenagem cirúrgica, para tumoração retroperitoneal. Após ressecção da massa, o paciente obteve melhora clínica, em uso de cetoconazol. A análise histopatológica evidenciou fenômeno de Splendore-Hoeppli, sugerindo infecção por Basidiobolus ranarum, uma zigomicose geralmente restrita ao tecido subcutâneo, com raro comprometimento gastrintestinal.

Entomophthoromycosis in South Bengal (Eastern India): a 9 years study.

Subcutaneous mycoses caused by the family Entomophthoraceae is very rare type of disease and is being reported sporadically from various Tropical countries including India. Here we report 8 cases of rhinoentomophthoromycosis caused by Conidiobolous coronatus and 7 cases of chronic subcutaneous phycomycosis caused by Basidiobolus ranarum. Cases were detected during a span of 9 years between 1991 to 1999, from 9 districts in and around Kolkata (Eastern India). Former type of lesions were detected among 20 to 65 age group of healthy individuals, predominantly males (7:1). In the latter type, male-female ratio was 2:5, and except for one all cases belonged to below 20 years age group of healthy individuals. Several cases were detected only after examination of repeat biopsy samples. With high degree of clinical suspicion, right approach is needed for laboratory confirmation of diagnosis.

Presumably entomophthoramycosis in an HIV-infected patient: the first in Thailand.

The authors reported the case of a symptomatic HIV-infected woman with a slowly progressive infiltrative lesion which invaded in and around the nasal cavity over a 6-month period. Physical examination showed erythematous to violaceous plaques at the nasal and malar areas. Swelling of the inferior turbinate was noted in the right nare. Skin biopsy of the involved area revealed multiple nonseptate, broad, thin-walled hyphae within giant cells and granulomata. Entomophthoramycosis was diagnosed based on clinical features and histopathology. She was treated with intravenous amphotericin B for two weeks, followed by oral itraconazole 400 mg daily. At six months there was complete resolution of all lesions.