Abstract
“Pébrine” is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.
Highlights
Pebrine disease of the silkworm Bombyx mori was first identified in France in 1845 and was the reason for the collapse of the French seri culture industry by 1865 (James and Li, 2012)
We recently developed a broad-range real-time quantitative PCR assay for the Nosema β-tubulin gene based on the SYBR-green detection chemistry, which has been used successfully to detect generic Nosema in wild and commercial silkworms (Esvaran et al, 2019), without being able to distinguish between different species of Nosema
The specificity of the TaqMan assay was tested by analysing samples containing three other silkworm pathogens: B. mori bidensovirus (BmBDV), B. mori nucleopolyhedrosis virus (BmNPV), and B. bassiana (B. mori muscardine)
Summary
Pebrine disease of the silkworm Bombyx mori was first identified in France in 1845 and was the reason for the collapse of the French seri culture industry by 1865 (James and Li, 2012). The disease is highly contagious and can transmit horizontally and vertically. The eukaryotic microsporidian Nosema responsible for the disease is a highly specialised eukaryotic intracellular parasite, highly adapted to the silkworm’s gut environment, where it germinates and penetrates the gut epithelium; acquiring host proteins for its survival and propagation (Li et al, 2018). Microsporidians generally infect the larval silkworms horizontally, through feeding, and if sufficient care is not taken the disease can be transmitted vertically from infected mother moth to its offspring (Hukuhara, 2018). It becomes increasingly important to screen the mother moths for the presence of N. bombycis spores
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