Research paper
New and rapid strategies for the diagnosis of bovine paratuberculosis “in situ” using latex particles

https://doi.org/10.1016/j.jim.2021.113085Get rights and content

Highlights

  • A visual immunoagglutination assay for the diagnosis of bovine paratuberculosis was developed.

  • The reagent is based on colored latex particles and a commercial paratuberculosis protoplasmic antigen.

  • The agglutination can be read after 5 min “in situ” in the herds and it is easy to implement.

  • The agglutination test may be a rapid and inexpensive diagnostic strategy to control PTB.

Abstract

The chemical coupling of a protoplasmatic antigen from Mycobacterium avium subsp. paratubeculosis onto core-shell carboxylated particles was investigated with the aim of producing latex-protein complexes to be used in immunoagglutination assays capable of detecting bovine paratuberculosis disease. For this purpose, sensitizations were carried out using both colored and not colored carboxylated latexes as well as the protoplasmatic antigen at pH close to its isoelectric point to favor the antigenic protein to approach the particle surface. In all cases, higher fractions of proteins were chemically-bound to carboxyl groups on the surface of the particles. The assessment of the performance of the visual immunoagglutination assays consisted of evaluating 111 sera from healthy and infected bovines with Mycobacterium avium subsp. paratuberculosis. Complexes obtained from the colored latex allowed an acceptable visual discrimination between the studied positive and negative sera. Most of the positive samples showed strong to very strong agglutination and only a few samples reacted weakly, i.e. a sensitivity of 70%. The specificity of the assay, on the other hand, was 86%. Therefore, this rapid detection technique allows an easy and inexpensive identification of animals possibly infected with paratuberculosis “in situ” in the herds.

Introduction

Paratuberculosis (PTB), or Johne's disease, is a chronic infectious granulomatous enteritis of ruminants, whose causative agent is Mycobacterium avium subsp. paratuberculosis (MAP) (Hermon-Taylor and Bull, 2002; Wadhwa et al., 2012; Patterson et al., 2020). In cattle, this disease produces persistent diarrhea, progressive weight loss, debilitation and, eventually, death (Collins, 2013; Martcheva et al., 2015; Alonso-Hearn et al., 2017). PTB is endemic worldwide, with high prevalence levels and public health relevance because of its possible association with Crohn's disease (Pickup, 2006; Ricchi, 2014; Waddell et al., 2016). In South America, PTB represents an important problem; in some regions of Argentina, particularly, the seroprevalence of this disease ranges from 7 to 19.6% (Espeschit et al., 2017).

This disease causes substantial economic losses to the cattle industry, mainly as the result of increased premature culling, replacement costs, decreased milk yield, reduced feed conversion efficiency, fertility problems, reduced slaughter values and increased susceptibility to other diseases (Ott et al., 1999; Weber, 2006; Gonda et al., 2007; Richardson and More, 2009). The global prevalence of PTB, along with the economic losses associated and its putative impact on trade have raised concern about this disease (Rideout et al., 2003; Spackman, 2011; Garcia and Shalloo, 2015).

MAP is excreted in large numbers in feces of infected animals and in lower numbers in their colostrum and milk (Cunha et al., 2020; Patel and Shah, 2011). This pathogen is resistant to environmental factors. It can survive on pasture for >1 year and its survival in water is longer than in soil (Whittington et al., 2004). The infection is usually acquired through the fecal-oral route: by ingestion of the organism when nursing on contaminated teats; consumption of milk, solid feed or contaminated water or by licking and grooming behavior in a contaminated environment. On the other hand, calves are the most susceptible to infection (Whittington et al., 2019).

Culture isolation of MAP from individual fecal samples is considered the reference test for PTB diagnosis (Clark et al., 2008; Arango-Sabogal et al., 2018). Considering the time, cost and difficulty of isolating MAP from feces, some alternative tests have become commercially available for PTB diagnosis. In this context, the detection of the organism in feces or tissue by PCR, immune response by skin testing, or interferon-γ and antibody by ELISA are the most used strategies in paratuberculosis control programs (Chaubey et al., 2016; Beaver et al., 2017; Navarro-Gonzalez et al., 2019).

Diagnostic methods play a crucial role in the correct identification of infected and disease-free animals, and serve as a basis for the implementation of control strategies and disease management programs. An alternative diagnostic method is the immunoagglutination assays (IA). Typical IA are based on latex microspheres with antigen (Ag) molecules bound to their surface (latex-protein complexes; LPC). In the immunoassay, an aqueous dispersion of these LPC are mixed with a sample containing antibody (Ab) molecules from whole blood or serum. The Ab molecules normally bind to Ag molecules situated on the surface of different micro/nanospheres and cause agglutination of latex particles. LPC production (a process called sensitization) requires that biomolecules can either be physically or chemically fixed to the surface of the polymer particles. The covalent coupling of proteins, unlike their physical adsorption, prevents the partial desorption of the bonded proteins and maintains their native conformation (Hidalgo-Alvarez and Galisteo-Gonzalez, 1995; Molina-Bolivar and Galisteo-Gonzalez, 2005). The main advantages of the latex agglutination test are its rapidness, simplicity, low cost and practicability (it can be visually observed).

In this research, we have developed an IA assay for the diagnosis (visual and “in situ”) of bovine PTB, by using both colored and not colored latex particles sensitized by covalent coupling of a protoplasmatic antigen (PPA) from MAP.

Section snippets

Materials and methods

Reagents: All chemicals employed in this study were of analytical grade and were used without further purification. Double-distilled and deionised water was utilized in all experiments. The synthesis of latex particles included Styrene (St) monomer (Pampa Energía S.A., Argentina), methacrylic acid (MAA, Merck), glycidyl methacrylate (GMA, Sigma-Aldrich), fuchsine (BF, Cicarelli) as well as potassium persulfate (KPS, K2S2O8, Mallinckrodt), as initiator, and dihexylsodium sulfosuccinate (MA80,

Synthesis and characterization of latex particles

An analysis of the main characteristics of the obtained latex particles revealed that, in all cases, final latex particles were quasi-monodisperse in size, adequately stable and suitable for their use in immunodiagnostic (Table 1). Both visual and DLS determinations proved that no coagulation occurred, even at the highest KBr concentration evaluated in the study. Poor coagulation tendency, which could be explained by the stabilizing effect of COOH groups on the surface of the particles, is a

Discussion and conclusions

In Argentina, the current reference detection test for PTB is MAP identification from bacterial culture. This technique, however, presents several disadvantages. First, it requires long incubation periods because of the intermittent excretion of MAP. This excretion pattern makes it advisable to take serial samples over time (Pinedo et al., 2008), which causes an epidemiologically dangerous delay in taking control measures. In addition, bacterial culture is too expensive to be routinely applied

Fundings sources

This work was partially supported by grants from INTA Castelar, Instituto de Biotecnología and PID 2015-061 (Dra Romano M.I.) awarded by contract between the Agencia de Promoción Científica y Tecnológica, INTA and the dairy cooperative La América de Suardi, Santa Fe.

Declaration of Competing Interest

The authors declare no potential conflicts of interest.

Acknowlegments

We thank Dra. Julia Sabio, from INTA-IABIMO, for the critical reading of the manuscript as well as CONICET, ANPCyT and the Universidad Nacional del Litoral for their financial support.

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