Gold nanoparticle based immunochromatographic biosensor for rapid diagnosis of Mycobacterium avium subspecies paratuberculosis infection using recombinant protein

https://doi.org/10.1016/j.mimet.2020.106024Get rights and content

Highlights

  • A gold nanoparticle based LFA using MAP recombinant protein (44 kDa) has been developed for sensitive and specific detection of PTB in field conditions.

  • The diagnostic sensitivity and specificity of the LFA for MAP specific antibodies was found approximately 84.2% and 83.3% in comparison to indirect ELISA.

  • MAP recombinant protein (44 kDa) based LFA would be an effective, easy to use, rapid, cost effective and on-site diagnostic test

Abstract

Highly infectious and obvious withstand ability of Mycobacterium avium subspecies paratuberculosis (MAP) to environment as well as lack of on-site field diagnostic methods notably hampers the paratuberculosis (PTB) control. The existing intricacy, time-consuming and complicated diagnostic methods of PTB accentuate the development of novel and easy-to-perform on-site test. A gold nanoparticle (GNP) based lateral-flow assay (LFA) using MAP recombinant protein (44 kDa) has been developed for sensitive and specific detection of PTB in field conditions. The diagnostic sensitivity and specificity of the LFA for MAP specific antibodies was found approximately 84.2% and 83.3% in comparison to indirect enzyme-linked immunosorbent assay. Consequently, the newly developed GNP based LFA offers on-site and cost-effective method for the prompt diagnosis of PTB and precludes the time-consuming laboratory screening.

Introduction

Mycobacterium avium subspecies paratuberculosis (MAP) implicated in chronic and contagious paratuberculosis (PTB) infection in ruminants as well as captive and free-ranging wild animals accounted for 200 million dollar loss in dairy sector of the US (Losinger, 2006). The clinical complications viz. granulomatous enteritis, persistent diarrhoea, progressive weight loss and debilitation frequently observed during PTB infection (Manning and Collins, 2001). Because of the global presence of PTB, it is frequent screened during International trade consequence to the mass culling of positive animals (Fecteau, 2018). Prevalence of PTB was maximum in African continent (53%) in comparison to other continents i.e. Europe (20%), Australia (6.8%), North America (16.9%), South America (18.3%) and India (23.3%) (Agrawal et al., 2019). On the basis of restriction fragment length polymorphisms (RFLP) analysis, MAP has been characterized into two main host-adapted types: type I or S strains found in sheep and type II or C strains found in cattle (de Juan et al., 2006). Young one's are highly susceptible as the maximum transmission of MAP occurs through milk (Steuer et al., 2020).

The diagnosis of disease is complicated and challenging in pre-clinical stage (i.e. infected animals do not exemplify any symptom of disease at an early stage) (Sergeant, 2003). At present, routine diagnosis of PTB is made at several laboratories by enzyme-linked immunosorbent assay (ELISA), skin testing for delayed type hypersensitivity, bacterial isolation, PCR and reverse transcriptase PCR (RT-PCR) (Reichel et al., 1999; Clark Jr et al., 2006; Shin et al., 2008; Weber et al., 2008; Thakur et al., 2019; Kalis et al., 2003; Cook et al., 2010;Eamens et al., 2000; Brey et al., 2006; Cook and Britt, 2007; Slana et al., 2008; Jaravata et al., 2006; Sonawane and Tripathi, 2019; Herthnek and Bölske, 2006). However, most of these diagnostic methods require the availability of a dedicated laboratory facility, highly trained laboratory personnel, stable reagents and multistep sample handling or preparation. In particular, MAP isolation requires well-established laboratory facility, which can be difficult and expensive to maintain, besides requiring 5 weeks to 6 months for the appearance of primary colonies on solid media. PCR test is based on actual DNA of organism, which is irrelevant in case of MAP, as sometime; there is no manifestation of disease despite organism present in body.

A rapid and easy-to-perform test, which would allow for on-site diagnosis to be made in the case of a suspected disease would circumvent issues associated with the transportation of samples to the laboratory and would be especially useful for a faster diagnosis in areas where the disease is endemic. When the disease occurs in a remote area which is poorly connected with road and railways, the requirement for a pen-side diagnostic test that is quick and easy to perform, devoid of sophisticated equipment or per se laboratory expertise will be highly valuable. For these reasons, a recombinant protein based lateral-flow assay (LFA) for the detection of PTB specific antibodies might be a key to overcome the issues associated with other assays and become an effective tools to diagnose PTB. The detection of PTB specific antibodies by direct application of clinical sample from animals of an infected farm may reduce the chances of diagnostic error arising from nonspecific reactions. The assay may be used to quickly diagnose the disease and could be an effective tool in controlling PTB.

Section snippets

Materials and methods

The MAP2963 recombinant protein (44 kDa) expression clone and the hyperimmune serum raised in guinea pig against MAP2963 recombinant protein (44 kDa) were gifted by Dr. P.P. Goswami (Head, Division of Veterinary Biotechnology, IVRI, Izatnagar, Bareilly (U.P.) India.

Characterization of bare and conjugated gold nanoparticles (GNPs) by UV- visible spectrophotometry and zeta sizer

`The absorption spectrum of synthesized bare 0.2 mM GNPs was measured from 450 nm to 600 nm and maximum absorbance (λmax) found at 520 nm was 0.599 (Fig. 2A). Similarly, the GNPs conjugated secondary antibody (anti-guinea pig/anti-bovine) absorption spectra were also measured from 450 nm to 600 nm and compared with bare GNPs absorption spectra, indicated the shifting of λmax from 520 nm to 522 nm (Fig. 2B and C). With an increase in the particle size of GNPs, the shifts in absorption peak

Discussion

`An indispensable part of any disease control strategy includes the deployment of diagnostic assays to rapidly confirm the initial clinical determination of infection. This is of particular importance because of the highly infectious nature of MAP, its fecal shedding during the prolong subclinical period or prior to any obvious clinical signs and remarkable withstand ability to environment. PTB differential diagnosis from parasitic infestations is a herculean task. Although bacteriological

Conclusion

In this study, the lateral flow assay against M. paratuberculosis was developed and diagnosis suitability of the assay was determined by using ELISA tested M. paratuberculosis positive and negative serum samples (n = 31). The results showed that the detection efficiency of lateral flow assay strip test against M. paratuberculosis was utmost similar as that of ELISA, an intricacy method. The overall sensitivity, specificity and PPV of the LFA were 84.2%, 83.3% and 88.89% respectively in

Authors' contributions

AA designed the study, carried out the experiment, analysed the data and wrote the manuscript. RV, AG, PK, MHK, RS, SK, and SKP assisted in research work, data curation and edited the manuscript. PS designed this study, analyzed the data and helped in research work.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgment

We are thankful to Dr. P. P. Goswami (Head, Division of Veterinary Biotechnology, IVRI, Izatnagar, Bareilly (U.P.) India for providing MAP Recombinant protein (44 kDa) and its hyperimmune sera raised in guinea pig and providing ELISA tested clinically infected MAP positive and negative serum samples. We are also thankful to IVRI for providing fund to this work.

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