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. 2026 Oct 13;12(6):e71278. doi: 10.1002/vms3.71278

Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Faeces Using a Rhodamine B Hydrazone Immunosensor on Antibody‐Coated Nitrocellulose Membranes

Mohammad Khosravi 1,✉, Darioush Gharibi 1, Rozhin Ansari 2, Mahdi Pourmahdi Borujeni 3, Mohammad Nouri 4
PMCID: PMC13660716  PMID: 42867473

ABSTRACT

Rapid, inexpensive and reliable detection of Mycobacterium avium subsp. paratuberculosis (MAP) is essential for the control of Johne's disease. This study developed a rhodamine B hydrazone nanoparticle‐based immunosensor coupled with antibody‐coated nitrocellulose membranes (IMS‐CNM) for the detection of MAP in bovine faecal samples. Anti‐MAP antibodies were purified from hyperimmune donkey and rabbit sera. Purified donkey IgG was immobilized on nitrocellulose membranes as the capture antibody, whereas rabbit anti‐MAP IgG was conjugated to rhodamine B hydrazone nanoparticles using EDC‐NHS chemistry to prepare the chromogenic immunosensor. The diagnostic performance of the assay was evaluated using 89 bovine faecal samples and compared with nested PCR. Nested PCR identified 62 positive and 27 negative samples, whereas the IMS‐CNM assay detected 60 positive and 29 negative samples. Using nested PCR as the reference method, the IMS‐CNM assay achieved a sensitivity of 90.32% (95% CI: 82.96%–97.68%) and a specificity of 85.19% (95% CI: 71.80%–98.58%). The analytical limit of detection was approximately 3 × 103 MAP cells/mL in PBS. No significant difference was observed between the IMS‐CNM assay and nested PCR in classifying positive and negative samples. The proposed IMS‐CNM assay provides a rapid, simple and cost‐effective approach for detecting MAP in bovine faeces with diagnostic performance comparable to nested PCR. Following further optimization and validation in larger field studies, this platform may serve as a practical screening tool for Johne's disease, particularly in resource‐limited settings.

Keywords: cattle, faeces, immunosensor, Mycobacterium avium subspecies paratuberculosis , rhodamine hydrazine

A rhodamine B hydrazone nanoparticle‐based immunosensor coupled with an antibody‐coated nitrocellulose membrane (IMS‐CNM) was developed for rapid detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine faeces. The assay combines antibody‐mediated bacterial capture with chromogenic nanoparticle detection, providing a simple, rapid and cost‐effective platform with diagnostic performance comparable to nested PCR.

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1. Introduction

Rapid and accurate detection of pathogenic microorganisms remains a major challenge in both veterinary and human medicine. Clinical diagnostic laboratories use a variety of methods to identify infectious agents, including microbial culture, colony counting, biochemical assays, serological tests and molecular techniques (Leonard et al. 2018). The culture‐based methods are often labour‐intensive, time‐consuming and require specialized laboratory facilities and trained personnel. Moreover, some pathogens are difficult or impossible to culture, limiting the effectiveness of conventional diagnostic approaches and delaying disease control measures (Luo et al. 2017; Landa et al. 2021). Consequently, molecular diagnostics and antibody‐based detection methods have become increasingly important for the rapid identification and screening of infectious diseases. Continuous advances in biosensor technology have further expanded the development of rapid, sensitive and user‐friendly diagnostic platforms that overcome many of the limitations of traditional techniques (Chaubey et al. 2016).

An ideal diagnostic assay should provide rapid, accurate and cost‐effective detection while minimizing procedural complexity and the need for sophisticated equipment. Antibodies remain among the most widely used recognition molecules because of their high specificity and affinity for target antigens. Rhodamine B derivatives have attracted considerable attention in biosensor development owing to their unique chromogenic and fluorescent properties. These compounds exhibit favourable characteristics, including high sensitivity, good water solubility, photostability and excellent optical performance (Wang et al. 2014). The spirolactam ring of rhodamine hydrazone undergoes structural opening in the presence of specific metal ions, such as Cu2+, resulting in a distinct pink colour accompanied by fluorescence emission (Sun et al. 2011). This optical response has been widely exploited in colourimetric and fluorescence‐based biosensors (Meng et al. 2013; Fang et al. 2015). Previously, Khosravi et al. (2021) developed a rhodamine hydrazone nanoparticle‐based immunosensor capable of detecting Mycobacterium avium subsp. paratuberculosis (MAP) through the intensity of the pink colour generated after binding of a rhodamine‐labelled antibody to MAP cells captured by iron nanoparticles.

Johne's disease, caused by MAP, is a chronic enteric disease that causes substantial economic losses in the livestock industry, particularly in dairy cattle. MAP is a slow‐growing, Gram‐positive, non‐spore‐forming and non‐capsulated bacterium. Infected animals may shed approximately 106–108 colony‐forming units (CFU) of MAP per gram of faeces (Rani et al. 2019), and ingestion of as few as 1.5 × 106 organisms can establish infection in calves (Sweeney et al. 2006). In addition to faecal–oral transmission, MAP may be transmitted through milk, colostrum and in utero. Increasing evidence also suggests a potential zoonotic role for MAP, as viable organisms have been detected in milk and isolated from patients with gastrointestinal disorders. Therefore, early and reliable identification of infected animals is essential for effective disease control and may also contribute to reducing potential public health risks (Kuenstner and Kuenstner 2021). Accordingly, considerable efforts have been devoted to developing sensitive and practical methods for MAP detection (Aydemir et al. 2023).

Polymerase chain reaction (PCR), particularly assays targeting the IS900 sequence, is widely used for the detection of MAP and has demonstrated high sensitivity for identifying subclinical infections (Münster et al. 2011). However, PCR performance is strongly influenced by inhibitory substances present in clinical specimens, especially faecal and milk samples, which may substantially reduce diagnostic sensitivity. Consequently, efficient DNA extraction remains a critical prerequisite for reliable molecular detection. Several PCR assays have been developed based on MAP‐specific genetic targets, including IS900, F57, hspX, ISMav2 and ISMap04 (Slana et al. 2008).

Previously, we developed an immunomagnetic bead–immunosensor (IMB‐IMS) platform for detecting MAP in milk, colostrum and faecal samples obtained from experimentally and naturally infected cattle (Khosravi et al. 2021, 2025). Although this method demonstrated high diagnostic performance, it requires multiple washing steps during immunomagnetic separation, making the procedure relatively time‐consuming and increasing the risk of losing captured bacteria or immunomagnetic beads. These limitations reduce its suitability for large‐scale field screening. Therefore, the present study aimed to simplify the detection procedure by immobilizing capture antibodies directly onto a nitrocellulose membrane (CNM), allowing MAP cells in faecal suspensions to bind directly to the membrane before detection with the rhodamine hydrazone immunosensor. This membrane‐based platform minimizes washing steps, shortens assay time, reduces cost and provides a practical approach for rapid, high‐throughput screening of MAP under field conditions.

2. Materials and Methods

2.1. Sample Collection

A total of 89 rectal faecal samples were collected from cattle in a commercial dairy herd undergoing routine annual screening for MAP using a commercial indirect ELISA kit (ID.VET, France) in accordance with the manufacturer's instructions. Based on the routine herd screening results, animals were classified as ELISA‐positive or ELISA‐negative. Animals that tested positive by the routine ELISA screening were separated from the healthy herd before the onset of clinical signs; therefore, the study population consisted of subclinical animals. In the current study, 89 cattle aged 6–24 months were selected from the screened herd, including 58 ELISA‐positive and 31 ELISA‐negative animals. Animals were selected from both ELISA categories to provide faecal samples representing the range of infection within the herd. Approximately 10 mL of faecal material was collected directly from the rectum of each selected animal using standard hygienic procedures and stored at −20°C until further analysis by nested PCR and the IMS‐CNM assay.

2.2. Preparation of Bacterial Strains

MAP strains III and V, together with Mycobacterium phlei, were confirmed by nested PCR targeting the IS900 sequence. The bacterial strains were cultured in Difco‐Henle medium at 37°C for 12 weeks. The cultures were harvested, washed twice with sterile phosphate‐buffered saline (PBS) by centrifugation for 15 min, and resuspended in PBS to the original volume. The bacterial suspensions were then adjusted to a turbidity equivalent to McFarland standard 2 and inactivated with 10% formalin for 24 h, as previously described by Chui et al. (2004). The inactivated bacterial suspensions were stored at −20°C until use.

2.3. Preparation of MAP‐Specific Antibodies

The concentration of formalin‐killed MAP was adjusted to 6 × 108 cells/mL according to the McFarland standard and mixed with an equal volume of ISA 70 adjuvant. Four rabbits were immunized by intramuscular and subcutaneous injection of 1 mL of the antigen suspension, while one donkey received 2 mL of the same preparation. Booster immunizations were administered at a concentration of 3 × 108 cells/mL emulsified with an equal volume of ISA 70 adjuvant. A total of four booster injections were given at 2‐week intervals.

One week after the final immunization, blood samples were collected from the hyperimmunized rabbits and donkey. The samples were allowed to clot at room temperature for 2 h and centrifuged at 1800 × g for 15 min. The sera were collected and stored at −20°C until use.

Capture and detector antibodies were purified from the hyperimmune sera by ion‐exchange chromatography according to Hay and Westwood (2002). Briefly, hyperimmune sera were precipitated with 33% saturated ammonium sulphate, and the precipitated proteins were collected by centrifugation at 400 × g for 15 min. After dialysis against 0.02 M phosphate buffer (PB, pH 7.2) for 48 h, IgG was purified using a DEAE‐cellulose column. The resin was equilibrated with 0.02 M PB (pH 7.2) for 24 h before packing into the column. The dialysed protein solution was applied to the column, incubated for 2 h after closing the outlet to facilitate binding and the IgG fraction was eluted with 0.02 M PB (pH 7.2).

MAP‐specific antibodies were subsequently purified by affinity chromatography as previously described by Khosravi et al. (2025). Briefly, formalin‐killed MAP cells were sonicated, and 5 mg of the resulting antigen was covalently coupled to CNBr‐activated Sepharose 4B (4B200, Sigma‐Aldrich). Total IgG was applied to the affinity column, and MAP‐specific antibodies were eluted with glycine buffer (pH 2.5). The eluate was immediately neutralized with 0.1 M glycine buffer (pH 9.5).

To reduce cross‐reactivity, the purified antibodies were adsorbed with M. phlei (McFarland 0.5) by incubation at room temperature for 1 h. The bacterial cells together with bound cross‐reactive antibodies were removed by centrifugation at 4000 × g for 10 min, and the resulting supernatant was collected as the anti‐MAP antibody preparation. The protein concentration and purity of the purified antibodies were determined using the Bradford assay and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE), respectively.

2.4. Evaluation of Antibody Titres Against MAP

The titres of hyperimmune sera and purified antibodies obtained from immunized rabbits and the donkey were evaluated by indirect ELISA. Briefly, sonicated MAP antigen was prepared as previously described (Khosravi et al. 2025) and diluted in carbonate–bicarbonate coating buffer (pH 9.6). A total of 1 µg of antigen was added to each well of a 96‐well ELISA plate and incubated overnight at 4°C. The wells were then blocked with 250 µL of 4% skim milk.

Hyperimmune sera were tested at dilutions of 1:20, 1:40, 1:80 and 1:100, whereas purified IgG samples were used at a concentration of 20 µg/mL. After incubation for 60 min, the plates were washed three times with PBS containing 0.05% Tween‐20 (PBST). Goat anti‐rabbit IgG conjugated to horseradish peroxidase (HRP) (ImmunoChemistry Technologies, USA; Cat. No. 6293) diluted 1:20,000 or HRP‐conjugated Protein A/G (Raha Azma, Iran) diluted 1:1000 was added for the detection of rabbit and donkey antibodies, respectively. Following incubation and three additional washes, 50 µL of tetramethylbenzidine (TMB) substrate was added to each well. The reaction was stopped after 5 min with 50 µL of 2% HCl, and the absorbance was measured at 450 nm using a microplate reader.

The specificity of the prepared hyperimmune sera (1:50 dilution) and purified antibodies (20 µg/mL) was evaluated against MAP, M. phlei, M. avium and Mycobacterium bovis using the same ELISA protocol. The analysis was performed before and after adsorption of cross‐reactive antibodies with either whole‐cell or sonicated M. phlei. Commercial M. avium and M. bovis tuberculin antigens (Razi Vaccine and Serum Research Institute, Karaj, Iran) were dialysed against PBS for 24 h to remove preservative additives before use as coating antigens.

2.5. Production of Rhodamine B Hydrazone and Immunosensor

Rhodamine B hydrazone was synthesized according to the methods described by Horzum et al. (2016) and Rai et al. (2018), with minor modifications. Briefly, 165 mg of rhodamine B (Merck, Cat. No. K13077599) was dissolved in 5 mL of ethanol and stirred for 15 min. Subsequently, 850 µL of monohydrazine (Sigma‐Aldrich, Cat. No. 207942) was added, and the reaction mixture was stirred for 6 h before being incubated overnight at 4°C. Thereafter, 10 mL of 1 M hydrochloric acid (HCl) was added, and the pH was adjusted to 10 using 1 M sodium hydroxide (NaOH). The resulting rhodamine B hydrazide precipitate was collected and washed three times with distilled water.

The purified rhodamine B hydrazide was dissolved in 5 mL of 1 mM ethanolic solution containing 0.5 M sucrose and heated to 90°C. Subsequently, 10 µL of N‐octylamine and 50 µL of 1 mM 3‐allylsalicylaldehyde (Sigma‐Aldrich, Cat. No. 636339) were added, and the reaction mixture was stirred at room temperature for 6 h. Formation of a characteristic pink precipitate confirmed the synthesis of rhodamine B hydrazone (Rai et al. 2018).

The immunosensor was prepared by conjugating rabbit anti‐MAP IgG to rhodamine B hydrazone using EDC‐NHS chemistry, as previously described by Khosravi et al. (2021). The conjugated immunosensor was blocked with 100 mM Tris‐HCl buffer (pH 8.8), followed by the addition of 10 µL of 5% Triton X‐100. After centrifugation at 8000 × g for 10 min, the supernatant was discarded, and the immunosensor pellet was resuspended in sterile PBS containing 1% bovine serum albumin (BSA). The final immunosensor preparation was stored at 4°C for up to 4 weeks.

2.6. Fourier‐Transform Infrared Spectroscopy

Antibodies, rhodamine B hydrazone and the prepared immunosensor were dried at 42°C for 72 h before analysis. The chemical structures of the individual components and the immunosensor conjugate were characterized by Fourier‐transform infrared (FTIR) spectroscopy (PerkinElmer 2400) using the potassium bromide (KBr) pellet method. Spectra were recorded over the range of 4000–400 cm−1.

2.7. Field Emission Scanning Electron Microscopy

The morphology of rhodamine B hydrazone, the immunosensor and antibody‐coated nitrocellulose membranes following reaction with faecal samples from healthy and MAP‐infected animals was examined by field emission scanning electron microscopy (FESEM). Samples were fixed with formaldehyde, dehydrated through a graded ethanol series, dried at 42°C for 72 h on aluminium foil, and analysed using a FESEM instrument.

2.8. Examination of Faecal Samples by PCR

Genomic DNA was extracted from bovine faecal samples using a commercial DNA extraction kit (CinnaClone, Iran) according to the manufacturer's instructions. Prior to extraction, frozen samples were thawed at room temperature. Nested PCR targeting the MAP‐specific IS900 sequence was performed using primers described by Corti and Stephan (2002). Positive and negative controls were included in each PCR run, with genomic DNA extracted from MAP serving as the positive control and distilled water as the negative control.

PCR amplification was carried out as previously described by Khosravi et al. (2025). Each 20 µL reaction contained 10 µL of master mix (Amplicon, Germany), 1 µL of each primer, 3 µL of template DNA and 5 µL of distilled water. Amplification was performed in a thermocycler (Mastercycler Gradient, Eppendorf, Germany) under the following conditions: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 30 s and extension at 72°C for 30 s; followed by a final extension at 72°C for 7 min. For the second‐round PCR, 3 µL of the first‐round PCR product was used as the template under identical cycling conditions. Amplified products were separated by agarose gel electrophoresis containing Safe Stain (CinnaGen, Iran) and visualized alongside a 50 bp DNA ladder (CinnaGen, Iran). Band intensity was visually scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong).

2.9. Detection of MAP Using IMS‐CNM

Approximately 0.2 g of homogenized faeces was suspended in 10 mL of PBS in a 15 mL Falcon tube and mixed thoroughly to obtain a uniform suspension. After standing for 5 min, the supernatant was transferred to a 50 mL Falcon tube and adjusted to a final volume of 10 mL with PBS.

Nitrocellulose membranes (1 × 1 cm) were incubated in PBS (pH 8.0) containing 100 µg/mL of donkey anti‐MAP antibody for 12 h to immobilize the capture antibody. The membranes were subsequently blocked with 4% skim milk for 2 h, stored at 4°C overnight, and washed three times with PBS. Each membrane was then transferred to a well of a sterile 24‐well plate containing 1 mL of a 1:1 mixture of faecal suspension and PBS and incubated on a shaker for 1 h. After three washes with PBS, 50 µL of the optimized rhodamine B hydrazone immunosensor suspended in 1 mL of PBS was added, followed by incubation for an additional 1 h with gentle shaking. The membranes were then washed five times with PBS.

Colour development was initiated by adding 150 µL of a freshly prepared solution consisting of 50 µL of 0.1 M copper sulphate and 100 µL of ethanol. The chromogenic reaction was recorded after 5 min and visually scored on a 4‐point scale: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong).

For quantitative analysis, membrane images were analysed using ImageJ software. The reacted area of each membrane was selected, and signal intensity was measured. The quantitative intensity (QI) for each membrane was calculated using the following equation:

QI=meanintensityofnegativecontrols−intensityofthesamplemembranearea

The calculated values were multiplied by 1000 to facilitate data analysis. The diagnostic performance of the IMS‐CNM assay was evaluated by comparison with nested PCR, and its sensitivity and specificity were subsequently determined.

2.10. Determination of the Limit of Detection of IMS‐CNM

The analytical limit of detection (LOD) of the IMS‐CNM assay was determined using experimentally prepared MAP suspensions in sterile PBS containing a faecal matrix derived from pooled faecal samples of three healthy bovines, all of which tested negative by nested PCR and IMS‐CNM. Because MAP cells exhibited considerable clumping, the bacterial suspension was thoroughly vortexed and then allowed to settle for 5 min. The upper fraction, enriched with dispersed bacterial cells, was collected for subsequent analysis. Six 10‐fold serial dilutions were prepared from the initial suspension (OD600 = 0.135). The bacterial concentration in each dilution was estimated by centrifugation at 7000 rpm for 8 min, followed by resuspension of the pellet in 20 µL of PBS and Ziehl–Neelsen staining. The bacterial count in each dilution was determined by microscopic examination and counting of bacterial cells within 10% of the stained smear area. The same dilutions were subsequently evaluated using the IMS‐CNM assay as described above, along with two negative controls. The experiment was performed three times independently. The LOD was determined based on the successful detection of the minimum concentration in three independent replicates, using both visual assessment of the chromogenic reaction and quantitative analysis of membrane colour intensity with ImageJ software.

2.11. Data Analysis

Data were analysed using SPSS version 26 (IBM Corp., Armonk, NY, USA). Descriptive statistics were calculated for all variables, including the optical density of the rhodamine B hydrazone nanoparticle‐based immunosensor (values below 3 were considered negative), the visual chromogenic reaction on the nitrocellulose membrane (ranging from 0, negative, to 1–3, positive) and the band intensity of the PCR test (ranging from 0, negative, to 1–3, positive), which was used as the reference method. Differences between groups were analysed using the Wilcoxon signed‐rank test, whereas agreement between the IMS‐CNM assay and nested PCR was assessed using the McNemar test and Cohen's kappa coefficient. The optimal diagnostic cut‐off value was determined by receiver operating characteristic (ROC) curve analysis. The ROC curve was generated by evaluating the diagnostic performance of various OD thresholds against the nested‐PCR classification. The threshold was derived from the current dataset. The cut‐off that provided the best discrimination between nested‐PCR‐positive and nested‐PCR‐negative samples was selected (maximum sensitivity and specificity). A p value ≤ 0.05 was considered statistically significant.

3. Results

3.1. Purity and Specificity of Anti‐MAP Antibodies

The purity of the anti‐MAP antibodies was evaluated by SDS‐PAGE, which demonstrated successful purification of the IgG fraction (Figure 1A). The hyperimmune response and the antigen‐binding activity of the purified antibodies were further confirmed by indirect ELISA (Figure 1B).

FIGURE 1.

FIGURE 1

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Purity and reactivity of the prepared antibodies against MAP. (A) SDS‐PAGE evaluation of antibody purification from hyperimmune sera of rabbits and donkey; (1) purified rabbit antibody; (2) serum proteins without IgG; (3) ammonium sulphate‐precipitated proteins; (4 and 5) purified IgG from rabbit and donkey, respectively. (B) Reactivity of various dilutions of hyperimmune sera and purified antibodies against MAP using ELISA. (C) Cross‐reactivity of the hyperimmune sera and purified antibody with other Mycobacterium strains; (D) after adsorption of cross‐reactive antibodies using whole M. phlei; (E) after adsorption of cross‐reactive antibodies using sonicated M. phlei antigens.

The cross‐reactivity of hyperimmune sera and purified antibodies with M. phlei, M. avium and M. bovis, before and after adsorption with whole‐cell or sonicated M. phlei, is presented in Figure 1C–E. Comparable levels of cross‐reactive antibodies were observed in the hyperimmune sera and purified IgG obtained from both rabbits and the donkey before adsorption. Following adsorption with M. phlei, the purified rabbit and donkey antibodies, which were subsequently used for immunosensor preparation and as capture antibodies, respectively, showed no detectable cross‐reactivity with the tested mycobacterial species. In contrast, the corresponding hyperimmune sera retained measurable cross‐reactivity after the adsorption procedure.

3.2. Characterization of the Immunosensor

The FTIR spectra of rhodamine B hydrazone, the purified antibody and the immunosensor are shown in Figure 2. Rhodamine B hydrazone exhibited a characteristic absorption peak at approximately 1045 cm−1. In the immunosensor spectrum, a broad absorption band at 3200–3600 cm−1 corresponded to O─H/N─H stretching vibrations. Characteristic antibody amide bands were observed at 1650–1630 cm−1 (Amide I), 1550–1520 cm−1 (Amide II) and 1300–1230 cm−1 (Amide III), indicating successful conjugation of rhodamine B hydrazone to the antibody. Additional absorption bands at 1400–1600, 1700–1680, 3200–3300 and 1250–1150 cm−1 were assigned to C═C, C═O, N─H and C─N functional groups, respectively, consistent with the chemical structure of rhodamine B hydrazone.

FIGURE 2.

FIGURE 2

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FTIR analysis of the binding of rhodamine hydrazone nanoparticles to the IgG antibody. The peaks located at 1650–1630, 1550–1520 and 1300–1230 cm−1 indicate the amide bonding of rhodamine hydrazone nanoparticles to the antibody.

3.3. Electron Microscopic Evaluation of MAP Capture on Nitrocellulose Membranes

FESEM demonstrated that both rhodamine B hydrazone nanoparticles and the antibody‐conjugated immunosensor nanoparticles exhibited a predominantly spherical morphology, with mean diameters of 86.16 ± 11 and 112.8 ± 13 nm, respectively (Figure 3A–D).

FIGURE 3.

FIGURE 3

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Electron microscopy images of rhodamine hydrazone nanoparticles and the immunosensor. (A, B) The rhodamine hydrazone nanoparticles (black arrows); (C, D) the rhodamine hydrazone immunosensor nanoparticles (black arrows).

As shown in Figure 4A,B, MAP cells were successfully captured on the antibody‐coated nitrocellulose membranes. Following incubation with the rhodamine B hydrazone immunosensor, abundant immunosensor particles were observed bound to the surface of the captured MAP cells (Figure 4C,D). In contrast, only minimal nonspecific deposition of the immunosensor was detected on the membranes of the negative control samples (Figure 4E,F).

FIGURE 4.

FIGURE 4

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Electron microscopy images of MAP captured on nitrocellulose membranes and detected using IMS. (A, B) The nitrocellulose membranes treated with cattle faeces infected with MAP. The aggregation of MAP on the absorbent membrane is visible in these images (red arrows). (C, D) The nitrocellulose membranes treated with cattle faeces infected with MAP bacteria and the IMS (yellow arrows). (E, F) The nitrocellulose membranes treated with cattle faeces free of MAP. The slight nonspecific binding of the IMS is shown in Panels E and F (yellow arrows).

3.4. Detection of MAP in Bovine Faecal Samples

The diagnostic performance of the IMS‐CNM assay was evaluated using nested PCR as the reference method. The results of PCR and visual IMS‐CNM grading are summarized in Table 1 and Figures S1 and S2. Among the 89 faecal samples analysed, nested PCR identified 62 (69.7%) positive and 27 (30.3%) negative samples, whereas the IMS‐CNM assay detected 60 (67.4%) positive and 29 (32.6%) negative samples. Based on PCR as the reference standard, the IMS‐CNM assay achieved a sensitivity of 90.32% (95% CI: 82.96%–97.68%) and a specificity of 85.19% (95% CI: 71.80%–98.58%). The mean visual colour intensity score was 1.09 ± 0.10 for the IMS‐CNM assay and 1.60 ± 0.14 for nested PCR (Figure 5A,B). The Wilcoxon signed‐rank test demonstrated a significant difference between the two methods (p ≤ 0.001).

TABLE 1.

Comparison of the results of PCR, ELISA and immunosensor on nitrocellulose membranes (IMS‐CNM) for detection of MAP in cow faecal samples.

PCR
Negative Positive Total
IMS‐CNM
Positive 4 56 60
Negative 23 6 29
Total 27 62 89
PCR
Negative Positive Total
ELISA
Positive 11 47 58
Negative 16 15 31
Total 27 62 89
ELISA
Negative Positive Total
IMS‐CNM
Positive 15 45 60
Negative 16 13 29
Total 31 58 89
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FIGURE 5.

FIGURE 5

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Detecting MAP using IMS‐CNM and PCR. (A) IMS‐CNM results are graded on a scale from 0 to 3. The absence of a visual signal in uninfected samples is represented by images labelled 0, while infection intensity levels are documented from 1 to 3. (B) PCR results are also graded on the same 0–3 scale. The intensity of the reactions is indicated above each image.

The ELISA identified 58 samples as positive and 31 as negative. Of the 62 PCR‐positive samples, 47 were correctly identified by ELISA, while 15 were classified as negative. Among the 27 PCR‐negative samples, 16 were correctly classified as negative and 11 were classified as positive. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of ELISA were 75.81% (95% CI: 63.85%–84.75%), 59.26% (95% CI: 40.73%–75.49%), 81.03%, 51.61% and 70.79%, respectively. Agreement between ELISA and PCR was fair (Cohen's κ = 0.337).

A direct comparison of the two immunological assays revealed that 45 samples tested positive by both IMS‐CNM and ELISA, while 16 samples tested negative by both methods. Fifteen samples were positive by IMS‐CNM but negative by ELISA, whereas 13 samples were negative by IMS‐CNM but positive by ELISA. Consequently, the overall agreement between IMS‐CNM and ELISA was 68.54%, with a Cohen's κ of 0.296, indicating fair agreement. McNemar's test showed no statistically significant difference in the discordant classifications between the two assays (p = 0.851).

ROC curve analysis yielded an area under the curve (AUC) of 0.90 (95% CI: 0.83–0.98), indicating promising diagnostic performance of the IMS‐CNM assay relative to nested PCR (Figure 6A,B). The McNemar test showed no statistically significant difference between the two diagnostic methods (p > 0.05), while Cohen's kappa coefficient demonstrated substantial agreement (κ = 0.74, p ≤ 0.001).

FIGURE 6.

FIGURE 6

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Comparison of the specificity and sensitivity of PCR and IMS‐CNM for the detection of MAP in faecal samples. (A) The ROC curve comparison of PCR and IMS‐CNM. (B) Comparison of PCR and IMS‐CNM results for detecting infected faeces according to pollution intensity.

Quantitative image analysis of the nitrocellulose membranes using ImageJ (Table S1) revealed a strong positive correlation between membrane QI and PCR results (Spearman's r = 0.68, p ≤ 0.001) and a very strong correlation between QI measurements and visual IMS‐CNM scores (Spearman's r = 0.94, p ≤ 0.001). Using PCR as the reference standard, the mean QI values were 2.37 ± 1.11 for PCR‐negative samples and 5.16 ± 2.26 for PCR‐positive samples. The Mann–Whitney test confirmed a significant difference in QI values between the two groups (p ≤ 0.001). The quantitative ImageJ analysis yielded the same sensitivity, specificity and AUC as the visual IMS‐CNM scoring system. The intraclass correlation coefficient (ICC) was 0.77 (95% CI: 0.64–0.85), indicating good agreement.

3.5. Analytical LOD of the IMS‐CNM Assay

The analytical LOD was evaluated using six 10‐fold serial dilutions of MAP suspended in sterile PBS containing a faecal matrix derived from pooled healthy bovine faecal samples. Positive reactions were observed in five out of the six serial dilutions. The lowest concentration producing a visible signal was approximately 3 × 103 cells/mL. Quantitative analysis of membrane colour intensity using ImageJ software showed results consistent with visual interpretation of the assay outcomes (Figure S3).

4. Discussion

Rapid and reliable detection of MAP remains a major challenge in veterinary diagnostics, particularly for large‐scale screening of subclinically infected animals. Although conventional methods, including bacterial culture, ELISA and PCR, are widely used, each has inherent limitations related to sensitivity, specificity, turnaround time or technical complexity (Janik‐Karpinska et al. 2022). PCR generally provides the highest analytical sensitivity, whereas antibody‐based assays often show lower sensitivity, especially when applied to milk samples. Consequently, only limited agreement has been reported between direct pathogen detection methods and indirect serological assays, although strong correlations are frequently observed among antibody‐based tests themselves.

In the present study, affinity‐purified anti‐MAP antibodies showed no detectable cross‐reactivity with M. phlei, M. avium or M. bovis after adsorption with M. phlei, whereas the corresponding hyperimmune sera retained measurable cross‐reactivity. This finding demonstrates that affinity purification combined with adsorption effectively improved antibody specificity. The persistence of cross‐reactive antibodies in the hyperimmune sera is likely attributable to antibodies recognizing conserved intracellular and surface antigens shared among mycobacterial species. Because capture of MAP in the IMS‐CNM assay depends primarily on the recognition of surface antigens, adsorption with whole M. phlei cells effectively removed antibodies directed against shared surface epitopes while preserving antibodies specific for MAP. Similar improvements in specificity following adsorption with M. phlei antigens have been reported previously, with serological specificity approaching 98% (Mikkelsen et al. 2011).

Previously, we developed an IMB‐IMS platform for MAP detection in milk, colostrum and faecal samples (Khosravi et al. 2021). In that study, immunomagnetic separation achieved a culture detection limit of approximately 3 CFU, and the combination of immunomagnetic capture with a rhodamine B hydrazone immunosensor enabled detection of approximately 30 cells. More recently, Khosravi et al. (2025) demonstrated that the IMB‐IMS assay showed diagnostic performance comparable to nested PCR for the detection of MAP in bovine faeces. Compared with the previously developed IMB‐IMS platform, the membrane‐based IMS‐CNM assay described in the present study demonstrated lower analytical sensitivity (LOD = 3000 cells); however, this detection limit is below the bacterial load shed by infectious animals (Rani et al. 2019). Nevertheless, replacing immunomagnetic separation with antibody‐coated nitrocellulose membranes substantially simplified the assay by eliminating the need for magnetic separation and multiple washing steps. These modifications reduced assay time, simplified the procedure and lowered the overall cost, making the method more suitable for routine screening of large numbers of samples under field conditions.

Although the visual intensity scores differed significantly between the IMS‐CNM assay and nested PCR, no significant difference was observed in their ability to classify samples as MAP‐positive or MAP‐negative. This finding suggests that the lower colour intensity observed with the membrane‐based assay mainly affects the estimation of bacterial load rather than the qualitative identification of infected animals. The substantial agreement between IMS‐CNM and nested PCR (κ = 0.74), together with the high sensitivity, specificity and area under the ROC curve, further supports the diagnostic potential of the proposed assay as a practical screening tool for Johne's disease. In addition, the IMS‐CNM assay demonstrated better agreement with nested PCR than ELISA, exhibiting higher sensitivity, specificity and overall accuracy. Notably, IMS‐CNM produced significantly fewer false‐positive and false‐negative results compared with ELISA. These findings suggest that the IMS‐CNM platform may offer a more reliable immunological approach for detecting MAP in bovine faecal samples, although PCR remains the reference method used in this study.

The present study has several practical advantages, including simplified sample processing, reduced assay cost and minimal equipment requirements. These characteristics make the IMS‐CNM platform particularly attractive for herd‐level surveillance and screening programmes, where rapid processing of large numbers of samples is essential. Nevertheless, further validation using larger populations, samples collected from multiple geographical regions and direct comparison with bacterial culture would strengthen the evidence supporting its routine diagnostic application.

As an alternative to conventional diagnostic methods, immunosensor‐based technologies have attracted increasing attention for the detection of pathogens and toxins. Owing to their rapid response, low sample volume requirements, cost‐effectiveness and ease of operation, immunosensors have emerged as promising tools for point‐of‐care and field diagnostics (Janik‐Karpinska et al. 2022). The IMS‐CNM platform developed in the present study extends this concept by combining antibody‐mediated capture on a nitrocellulose membrane with a rhodamine B hydrazone‐based chromogenic immunosensor. This approach provides a simple and rapid colourimetric assay that can be performed without specialized laboratory equipment, making it particularly suitable for large‐scale screening applications.

Dixit et al. (2023) reported only moderate agreement (κ = 0.571) between Ziehl–Neelsen staining and PCR for the detection of MAP in faecal samples from small ruminants. In that study, numerous Ziehl–Neelsen‐positive samples were PCR‐negative, whereas several PCR‐positive samples were not detected by microscopy, highlighting the limited diagnostic agreement between the two methods. In contrast, the IMS‐CNM assay developed in the present study showed substantial agreement with nested PCR (κ = 0.74), with no significant difference in the classification of positive and negative samples. These findings indicate that the proposed assay provides greater diagnostic consistency than conventional acid‐fast staining while maintaining the practical advantages of a simple colourimetric test.

Taddei et al. (2004) demonstrated that dot‐blot hybridization was more sensitive than agarose gel electrophoresis or a colourimetric detection kit for visualizing PCR products, particularly in samples containing higher bacterial loads. Similarly, the present study showed stronger agreement between IMS‐CNM colour intensity and PCR band intensity in samples with greater MAP contamination, suggesting that the chromogenic response reflects bacterial abundance more accurately at higher concentrations.

Recently, Aydemir et al. (2025) detected viable MAP in raw and pasteurized milk using propidium monoazide real‐time PCR and reported viable bacteria only in a small proportion of raw milk samples. Although the IMS‐CNM assay was developed as a diagnostic platform rather than a viability assay, its antibody‐based capture strategy may also have broader applications. The nitrocellulose membrane‐based separation approach described here could potentially be integrated with bacterial culture or molecular assays to improve MAP recovery from complex matrices such as milk, colostrum and faeces. Such integration may enhance the analytical sensitivity of downstream diagnostic methods while preserving the simplicity and low cost of the initial sample preparation step.

Several nanoparticle‐based biosensors have been developed for the rapid detection of MAP. Tallury et al. (2010) employed MAP‐specific antibodies together with ruthenium bipyridine (RuBpy)‐doped silica nanoparticle‐labelled antibodies to generate a fluorescent signal that enabled indirect immunofluorescence detection of MAP in bacterial mixtures and sputum samples. Although this fluorescence‐based assay successfully detected MAP within approximately 4 h after sample preparation, it required fluorescence microscopy and specialized instrumentation. In contrast, the IMS‐CNM assay developed in the present study provides a simple colourimetric readout that can be completed within approximately 3 h without the need for specialized detection equipment, making it more suitable for routine field screening.

Environmental surveillance has also been proposed as an effective strategy for monitoring Johne's disease at the herd level. Hahn et al. (2017) demonstrated that quantitative PCR analysis of boot swabs and liquid manure samples could reliably identify MAP‐contaminated dairy herds. Although environmental sampling provides valuable herd‐level information, it does not directly assess infection status in individual animals. In comparison, the IMS‐CNM assay was designed for direct detection of MAP in individual faecal samples, offering a practical alternative for animal‐level screening.

Jeyanathan et al. (2006) developed an in situ hybridization assay for detecting MAP in tissue samples from experimentally infected mice, reporting diagnostic performance comparable to Ziehl–Neelsen staining. Similarly, Kumanan et al. (2009) described a nucleic acid lateral‐flow assay based on DNA hybridization on polyethersulphone membranes for the detection of MAP RNA, with a detection limit of 10 CFU; however, the complete procedure required approximately 10 h. In contrast, the IMS‐CNM assay developed in the present study detected MAP directly in faecal samples without extensive sample processing. Moreover, its diagnostic sensitivity and specificity were comparable to those of nested PCR, with no statistically significant difference observed between the two methods. These findings indicate that the proposed assay combines rapid analysis with satisfactory diagnostic performance, supporting its potential application as a practical screening tool for Johne's disease.

Vitense et al. (2021) developed a gas chromatography‐based approach for detecting volatile organic compounds produced during MAP culture from faecal samples of infected ruminants and reported promising results for early bacterial detection. However, culture‐dependent approaches remain limited by the prolonged incubation time required for MAP growth, overgrowth by contaminating faecal microorganisms and the need for specialized analytical instrumentation to identify volatile metabolites. In contrast, the IMS‐CNM assay developed in the present study directly captures MAP from faecal samples using antibody‐coated nitrocellulose membranes and detects the bacteria with a rhodamine B hydrazone immunosensor. This approach substantially reduces the assay time while eliminating the need for bacterial culture, sophisticated instrumentation or highly specialized personnel.

Several studies have suggested that combining direct bacterial detection with serological testing improves the diagnosis of Johne's disease. Maroudam et al. (2015) and Garg et al. (2015) reported that the combined use of pathogen detection and antibody assays provided greater diagnostic accuracy than either method alone. Similarly, Park et al. (2020) observed that some infected animals intermittently shed MAP despite having no detectable antibody response, whereas animals with high antibody titres were more likely to be faecal‐positive. These findings highlight the complementary roles of faecal and serological testing in herd surveillance. Because the IMS‐CNM assay provides rapid detection of MAP directly from faecal samples, it could serve as a practical complementary test alongside routine serological screening, particularly for the early identification of infected animals.

Yakes et al. (2008) developed a surface‐enhanced Raman scattering (SERS)‐based assay for detecting MAP antigens from sonicated bacteria, achieving detection limits of 500 and 1000 cells/mL in experimentally contaminated PBS and milk samples, respectively. Although the analytical sensitivity of the SERS assay was high, it required Raman spectroscopy instrumentation and experimentally prepared samples. In contrast, the IMS‐CNM assay was evaluated using clinical faecal samples from naturally infected cattle, and its diagnostic performance was validated against nested PCR. These findings demonstrate that the proposed assay provides a practical balance between analytical performance, simplicity, rapid turnaround and cost‐effectiveness, supporting its potential use as a field‐deployable screening method for Johne's disease.

The IMS‐CNM assay developed in this study integrates bacterial capture and detection into a single workflow without requiring sophisticated instrumentation, specialized laboratory facilities or technically demanding procedures. Compared with conventional diagnostic approaches, such as PCR and ELISA, the assay can provide results within approximately 3 h while maintaining satisfactory diagnostic performance. Although the costs associated with antibody production, purification and conjugation are comparable to those of other immunoassays, the use of an inexpensive nitrocellulose membrane substantially reduces the overall assay cost, making the method suitable for laboratories with limited resources and potentially for on‐farm testing.

Despite these advantages, several aspects require further optimization before clinical implementation and commercialization. These include improving the long‐term stability of antibody‐coated membranes, enhancing the stability of the rhodamine B hydrazone immunosensor, minimizing nonspecific interactions between the immunosensor and the membrane, reducing the overall assay time and evaluating alternative membrane materials with greater mechanical stability and storage durability.

No single diagnostic assay is sufficient for the definitive diagnosis of Johne's disease, particularly during the early and subclinical stages of infection. Therefore, combining direct pathogen detection with serological testing is generally considered the most reliable diagnostic strategy. Owing to its rapid turnaround time, low cost and diagnostic performance comparable to nested PCR, the IMS‐CNM assay has the potential to serve as a practical screening tool or as a complementary test alongside established diagnostic methods. The present study has several limitations. First, diagnostic performance was evaluated using samples collected from a single dairy farm, which may limit the generalizability of the findings. Therefore, multicentre validation involving herds with different epidemiological backgrounds is required to confirm the reported sensitivity and specificity. Second, although substantial agreement was observed between the IMS‐CNM assay and nested PCR, the prevalence‐ and bias‐adjusted kappa (PABAK) value of 0.78 indicates that additional validation using a larger and more diverse sample population is warranted. Future studies should also evaluate the performance of the assay under routine field conditions and investigate its applicability to other clinically relevant sample types, including milk and colostrum. In addition, MAP exhibits a tendency to form bacterial aggregates under laboratory conditions. Despite efforts to use dispersed bacterial suspensions for spiking diluted faecal samples, the reported LOD was determined using MAP preparations in which some degree of bacterial clumping remained. This aggregation may interfere with accurate enumeration of MAP cells and could also affect efficient antigen capture on the capture membrane, potentially influencing the actual detection limit of the assay.

Selection and spectrum bias should be considered when interpreting the diagnostic performance of the IMS‐CNM assay. The animals included in this study were selected from a single commercial dairy herd and ranged in age from 6 to 24 months, with sampling based on their routine ELISA screening status. In addition, the study population predominantly consisted of subclinical animals and did not encompass the full spectrum of MAP infection, such as animals with advanced clinical disease or cattle from MAP‐free herds. Since diagnostic sensitivity and specificity may vary depending on the stage of infection, bacterial load and host characteristics, the performance estimates obtained in this study may differ from those observed in populations with a broader disease spectrum. Furthermore, because the ROC cut‐off was optimized using the same dataset on which diagnostic performance was subsequently evaluated, there is a possibility of optimism bias in the reported sensitivity and specificity. Therefore, the ROC‐derived cut‐off and corresponding diagnostic performance should be considered internally derived estimates, and independent validation using an external sample set is necessary to establish the generalizability of the cut‐off.

5. Conclusion

The present study successfully developed and validated a rhodamine B hydrazone‐based immunosensor coupled with an antibody‐coated nitrocellulose membrane (IMS‐CNM) for the rapid detection of MAP in bovine faecal samples. The assay demonstrated a lower LOD compared with previously reported rhodamine‐based immunosensors combined with immunomagnetic separation. However, the sensitivity and specificity of the IMS‐CNM assay showed no significant differences compared with nested PCR for the classification of MAP‐positive and MAP‐negative samples. Beyond its satisfactory diagnostic performance, the IMS‐CNM assay provides a simple, rapid and cost‐effective detection platform requiring minimal laboratory infrastructure, making it suitable for routine herd screening and application in resource‐limited settings compared with conventional and advanced diagnostic methods. Although further optimization is needed to enhance membrane stability, minimize nonspecific interactions and validate performance in larger and more diverse sample populations, the proposed platform represents a promising complementary tool for the diagnosis and surveillance of Johne's disease.

Author Contributions

Mohammad Khosravi: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, visualization, validation, methodology, project administration, formal analysis, resources, supervision. Darioush Gharibi: supervision, resources, methodology, validation, visualization, writing – review and editing, investigation, conceptualization, formal analysis, project administration. Rozhin Ansari: data curation, software, writing – original draft, investigation, visualization, writing – review and editing. Mehdi Pourmehdi Bourojeni: supervision, resources, data curation, software, formal analysis, validation, methodology, conceptualization, writing – review and editing. Mohammad Nouri: resources, supervision, conceptualization, methodology, writing – review and editing, project administration.

Funding

The authors gratefully acknowledge the financial support provided by Shahid Chamran University of Ahvaz, Ahvaz, Iran, under Grant No. SCU.VP1403.12470.

Ethics Statement

All animal experiments were conducted in accordance with the animal welfare regulations and the ethical guidelines of the Research Ethics Committee of Shahid Chamran University of Ahvaz. The study protocol was approved by the committee under the ethical approval code IR.SCU.REC.1402.018.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting file 1: vms371278‐sup‐0001‐SuppMat.docx

VMS3-12-e71278-s001.docx (825.3KB, docx)

Acknowledgements

The authors express their gratitude to the management and staff of commercial cattle farms in Isfahan, Iran, for their collaboration in furnishing clinical samples.

Data Availability Statement

All analysed/raw data are available on request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting file 1: vms371278‐sup‐0001‐SuppMat.docx

VMS3-12-e71278-s001.docx (825.3KB, docx)

Data Availability Statement

All analysed/raw data are available on request from the corresponding author.

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