Development and validation of real-time PCR for rapid detection of Mecistocirrus digitatus.

Subhra Subhadra,Mohanraj Karthik,Muthusamy Raman

doi:10.1371/journal.pone.0063019

Subhra Subhadra, Mohanraj Karthik + Show 1 more

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https://doi.org/10.1371/journal.pone.0063019

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Journal: PloS one	Publication Date: Apr 30, 2013
Citations: 19	License type: CC BY 4.0

Affiliation: Tamil Nadu Veterinary and Animal Sciences University

Abstract

Hematophagous activity of Mecistocirrus digitatus, which causes substantial blood and weight loss in large ruminants, is an emerging challenge due to the economic loss it brings to the livestock industry. Infected animals are treated with anthelmintic drugs, based on the identification of helminth species and the severity of infection; however, traditional methods such as microscopic identification and the counting of eggs for diagnosis and determination of level of infection are laborious, cumbersome and unreliable. To facilitate the detection of this parasite, a SYBR green-based real-time PCR was standardized and validated for the detection of M. digitatus infection in cattle and buffaloes. Oligonucleotides were designed to amplify partial Internal Transcribed Spacer (ITS)-1 sequence of M. digitatus. The specificity of the primers was confirmed by non-amplification of DNA extracted from other commonly occurring gastrointestinal nematodes in ruminants. Plasmids were ligated with partial ITS-1 sequence of M. digitatus, serially diluted (hundred fold) and used as standards in the real-time PCR assay. The quantification cycle (Cq) values were plotted against the standard DNA concentration to produce a standard curve. The assay was sensitive enough to detect one plasmid containing the M. digitatus DNA. Clinical application of this assay was validated by testing the DNA extracted from the faeces of naturally infected cattle (n = 40) and buffaloes (n = 25). The results were compared with our standard curve to calculate the quantity of M. digitatus in each faecal sample. The Cq value of the assay depicted a strong linear relationship with faecal DNA content, with a regression coefficient of 0.984 and efficiency of 99%. This assay has noteworthy advantages over the conventional methods of diagnosis because it is more specific, sensitive and reliable.

Highlights

Sub-clinical parasitism due to gastrointestinal nematodes (GINs) is a major problem in ruminant livestock because it leads to huge financial losses
M. digitatus may be found in mixed infection in ruminants along with other GINs such as Ostertagia ostertagi, Trichostrongylus spp., Haemonchus placei, Cooperia spp., etc., which makes the eradication of GINs a difficult task using chemotherapeutic agents [9]
Standardization of Conventional PCR Specific amplification of 182 bp was observed with the positive control at 58uC and 1.5 mM MgCl2, while the No-template control (NTC) did not show any amplification

Summary

Introduction

Sub-clinical parasitism due to gastrointestinal nematodes (GINs) is a major problem in ruminant livestock because it leads to huge financial losses. Among the GINs affecting large ruminants, Mecistocirrus digitatus, commonly known as a large stomach worm, is a bloodsucking helminth found in the abomasum of Asian zebu cattle (Bos indicus) and buffalo (Bubalis bubalis) [1]. Occurrence of M. digitatus was recorded as early as the 1920s, from the abomasum of cattle in India [3,4]. Various studies have confirmed the presence of M. digitatus infection in cattle and buffaloes in different parts of India [5,6,7,8]. The longer pre-patent period (up to 60 days) of this bloodsucking GIN [14] affects early detection

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