Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

Ravikumar, G.; Urs, S. Raje; Prakash, Narendra; Rao, C.G.P.; Vardhana, K.V.

doi:10.1016/j.jip.2011.04.009

Cited by 28 publications

(32 citation statements)

References 13 publications

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“…This improved method discriminates between N. apis and N. cerenae, has a detection limit of 100 spores per honeybee and lets us know the infection level (spore load) of samples. Similarly to other available molecular techniques, our real-time PCR-based method is as good as light microscopy analysis (Bourgeois et al 2010;Chaimanee et al 2011;Chen et al 2008;Hamiduzzaman et al 2010;Klee et al 2007;Martín-Hernández et al 2007;Ravikumar et al 2011;Stevanovic et al 2010;Traver and Fell 2011;Yoshiyama and Kimura 2011). Nevertheless, it does not rely on the expertise of the observer (Weiss et al 1999) and allows the complete (16S rRNA of N. apis, 16S rRNA of N. ceranae and ACTIN of A. mellifera) analysis of 10 samples in 3 h.…”

Section: Discussionmentioning

confidence: 98%

Nosema ceranae an emergent pathogen of Apis mellifera in Chile

2012

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The microsporidian Nosema apis and Nosema ceranae have been associated with colony disorders of Apis mellifera and Apis cerana, respectively. N. apis is endemic in South America. Recently, N. ceranae has been detected in Brazil, Uruguay and Argentina. No report of its presence, distribution and prevalence in Chile is available. Here, we present a real-time PCR-based method that was able to discriminate between N. apis and N. ceranae. The dynamic range of this assay was 100 to 100,000 spores per honeybee. False-negative results were avoided due to the use of ACTIN gene as internal standard. False-positive results were obtained neither in experimentally nor in naturally contaminated samples. Using this method, we screened 240 beehives from the Chilean region where 42% of the total country honey production take places (Región del Biobío). Nosema spp. were detected in the four provinces and in 20 of the 26 communes of the region. Among the samples analysed, 49% were positive for N. ceranae. Their infection level ranged from 200 to more than 100,000 spores per honeybee. N. apis was not detected in this region. Hence, our data show that in Chile N. ceranae is an emergent pathogen that is been replacing N. apis. Also, they support that N. ceranae maybe the actual responsible for nosemosis in A. mellifera in South America.

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Section: Discussionmentioning

confidence: 98%

Nosema ceranae an emergent pathogen of Apis mellifera in Chile

2012

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“…The silkworm rearing and microsporidian infection were essentially performed as reported by us (Ravikumar et al, 2011). Silkworms, B. mori (Pure Mysore) were fed on mulberry leaves.…”

Section: Silkworm and Microsporidian Infectionmentioning

confidence: 99%

“…DNA extracted from the mid gut of infected and control using Hi-Pure DNA extraction Kit (Himedia) according to manufacturer's protocol. DNA from mulberry leaves and pebrine infected A. mylitta DNA were used as reported earlier (Ravikumar et al, 2011). The DNA was analyzed in 1% agarose gel electrophoresis and quantifi ed using a Nanodrop (Thermo Corporation) spectrophotometer.…”

Section: Dna Extractionmentioning

confidence: 99%

“…This limitation has made it diffi cult to conduct investigations into microsporidian prevalence and transmission. To overcome these diffi culties, PCR-based techniques have been developed to detect the major pathogens of silkworms with great specifi city and sensitivity (Hatakeyama and Hayasaka 2003, Hamiduzzaman et al, 2010, Ravikumar et al, 2011, Fu et al, 2016.…”

Section: Introductionmentioning

confidence: 99%

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PCR-based detection of microsporidia in silkworms using non-conventional RNA polymerase primers

Roy¹,

Mandal²,

Ravikumar³

2017

Biosci. Biotech. Res. Comm

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Microsporidia are obligate intracellular, spore-forming parasites that infect both invertebrates and vertebrates. They infect silkworms causing the deadly pebrine disease leading to heavy crop loss in sericulture. Because of the horizontal and vertical transmittance, outbreaks should be detected at an early stage and persistent infections should also be identifi ed to prevent further transmittance. So far, microscopic examination method remains the conventional detection method for screening of microsporidia in sericulture. Molecular diagnosis tools have an advantage over microscopic detection as they are more specifi c, sensitive and aid in early detection. Microsporidia detection by PCR method using primers designed from SSU-rRNA is widely used. In this study, we developed a PCR assay for the detection of microsporidia using primers designed from the conserved regions of RNA polymerase gene. Under optimized PCR conditions, the assay yielded a ~650 bp DNA fragment from microsporidia infected silkworms, Bombyx mori and Antheraea mylitta. Sequence analysis of the amplifi ed products has shown homology to various microsporidia including Nosema bombycis and N. antheraea. No non-specifi c products were observed. This method could help in early detection of microsporidia infection at any developmental stage of the silkworm and thereby reducing the crop loss.

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“…The genome of N. bombycis was published in 2013, and studies have shown that the genome of N. bombycis is expanded, rather than compact as some other parasitic microsporidian genomes are through several common molecular mechanisms [12]. Other studies on N. bombycis have mainly focused on its classification [13,14], detection and control [15,16], as well as its metabolism [17,18]. There are only a few findings regarding the genes that play a role in the germination process of microsporidia.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome sequencing and characterization of ungerminated and germinated spores of <italic>Nosema bombycis</italic>

Liu

et al. 2016

ABBS

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Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori. Germination, an indispensible process through which microsporidia infect the host cells, is regarded as a key developmental turning point for microsporidia from dormant state to reproduction state. Thus, elucidating the transcriptome changes before and after germination is crucial for parasite control. However, the molecular basis of germination of microsporidia remains unknown. To investigate this germination process, the transcriptome of N. bombycis ungerminated spores and germinated spores were sequenced and analyzed. More than 60 million high-quality transcript reads were generated from these two groups using RNA-Seq technology. After assembly, 2756 and 2690 unigenes were identified, respectively, and subsequently annotated based on known proteins. After analysis of differentially expressed genes, 66 genes were identified to be differentially expressed (P ≤ 0.05) between these two groups. A protein phosphatase-associated gene was first identified to be significantly up-regulated as determined by RNA-Seq and immunoblot analysis, indicating that dephosphorylation might potentially contribute to microsporidia germination. The DEGs that encode proteins involved in glycometabolism, spore wall proteins and ricin B lectin of N. bombycis were also analyzed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed genes responsible for some specific biological functions and processes. The datasets generated in this study provide a basic characterization of the transcriptome changes in N. bombycis during germination. The analysis of transcriptome data and identification of certain functional genes which are robust candidate genes related to germination will help to provide a deep understanding of spore germination and invasion.

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Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

Cited by 28 publications

References 13 publications

Nosema ceranae an emergent pathogen of Apis mellifera in Chile

Nosema ceranae an emergent pathogen of Apis mellifera in Chile

PCR-based detection of microsporidia in silkworms using non-conventional RNA polymerase primers

Transcriptome sequencing and characterization of ungerminated and germinated spores of <italic>Nosema bombycis</italic>

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Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

Cited by 28 publications

References 13 publications

Nosema ceranae an emergent pathogen of Apis mellifera in Chile

Nosema ceranae an emergent pathogen of Apis mellifera in Chile

PCR-based detection of microsporidia in silkworms using non-conventional RNA polymerase primers

Transcriptome sequencing and characterization of ungerminated and germinated spores of &lt;italic&gt;Nosema bombycis&lt;/italic&gt;

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Transcriptome sequencing and characterization of ungerminated and germinated spores of <italic>Nosema bombycis</italic>