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. 2025 Apr 28;11(3):e70346. doi: 10.1002/vms3.70346

Assessing the Effectiveness of Immunoelectric Method in Detecting Mycobacterium avium Subspecies paratuberculosis in Cow Faeces With Paratuberculosis

Mohammad Khosravi 1,✉, MohammadRahim Haji Hajikolaei 2, Mohammad Nouri 2, Shayan Kalanter 3
PMCID: PMC12036691  PMID: 40294094

ABSTRACT

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in ruminants, is typically transmitted through the faecal–oral route. This study aimed to optimize and evaluate an immunoelectric (IE) method for detecting MAP in faecal samples from infected cows. The necessary polyclonal antibodies were extracted from hyperimmune donkeys and rabbits using affinity chromatography. Furthermore, cross‐reactive antibodies were eliminated through absorption with Mycobacterium phlei. The binding of donkey antibodies to a polystyrene filter and rabbit antibodies to Fe nanoparticles was facilitated by a diethylenetriaminepentaacetic acid (DTPA) linker. The trapping of bacteria on the filter and the fixation of Fe nanoparticles attached to specific antibodies led to a modification in the electrical resistance of the filter. This alteration in electrical resistance can be quantified using a high‐precision electrical meter. In this research, MAP was identified through both polymerase chain reaction (PCR) and the IE methods in faecal samples from dairy cows, producing varied outcomes in the enzyme‐linked immunosorbent assay (ELISA) test. The sensitivity and specificity of the ELISA approach for detection of the serum antibodies in comparison to the PCR technique were determined to be 75% and 72%, respectively. In contrast, the sensitivity and specificity of the IE method relative to the PCR approach were found to be 96% and 95%, respectively. With a detection time of less than 60 min, cost‐effectiveness per sample, user‐friendly operation utilizing an IE device, no requirement for specialized machinery and applicability in farm or dairy settings, this technique emerges as a promising alternative to traditional bacterial detection methods.

Keywords: bovine, faeces, immunoelectric, Mycobacterium avium subspecies paratuberculosis

This technique emerges as a promising alternative to traditional bacterial detection methods.

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1. Introduction

Johne's disease is a chronic bacterial infection that poses significant economic challenges to the dairy cattle industry. The causative agent, Mycobacterium avium subspecies paratuberculosis (MAP), is a gram‐positive bacterial pathogen characterized by its slow growth and absence of capsules and spores. To facilitate in vitro growth, culture media must be supplemented with mycobactin. Cattle exhibiting either the clinical or subclinical form of Johne's disease can shed the pathogen into the environment at levels ranging from 10 CFU/g faeces in light shedders to greater than 104 CFU/g faeces in super shedders. This shedding can facilitate the transmission of infection to young calves and other livestock (Crossley et al. 2005; Corbett et al. 2018). The infectious dose for young calves varies from 103 to 1.5 × 106 CFU (Sweeney et al. 2006; Whittington and Sergeant 2001).

Transmission can occur through several routes, including intrauterine transfer from placenta to foetus, as well as through milk and colostrum. The primary sources of MAP contamination on farms, pastures and livestock storage areas are the faeces of infected animals, which spread the bacteria among ruminants via contaminated food and water. Additionally, aerosol transmission is a potential route for the spread of Johne's disease, complicating control efforts (Eisenberg et al. 2010; Rodgers et al. 2007). A rapid and timely diagnosis of the underlying cause of a disease is crucial for implementing effective control measures. Management strategies aimed at reducing transmission among livestock can help mitigate the severity of the disease. These strategies include testing, culling, slaughter programmes and vaccination (Windsor 2015). Additionally, quarantine measures in animal husbandry, preventing the entry of suspicious animals and minimizing contact between calves and adult animals’ faeces are essential for decreasing disease incidence (Vilar et al. 2015). Currently, there is no effective drug treatment available for the disease, and treating infected animals is not recommended (Sweeney et al. 2012). The most effective approach to managing the disease is the elimination of infected livestock. Common methods for paratuberculosis diagnosis include the enzyme‐linked immunosorbent assay (ELISA) test, microscopic examination of faecal samples, polymerase chain reaction (PCR) and faecal culture, each with varying sensitivity and specificity (Constable et al. 2017; Nielsen et al. 2008). The ELISA test is employed for the detection and screening of antibodies in blood serum samples (Pruvot et al. 2013). The absorbent ELISA method has proven effective in diagnosing various diseases. Specific antibodies for MAP can be detected in both milk and blood samples. However, the sensitivity of the ELISA method varies depending on the stage of the disease in the affected cow (Nielsen et al. 2008).

Another diagnostic approach is based on interferon‐gamma testing, which utilizes the immune response. In this method, T lymphocytes are stimulated by exposure to various antigens, leading to the secretion of interferon‐gamma. It is important to note that the results can be difficult to interpret due to variations in the type and amount of the injected antigens, as well as the immune responses of the infected animals (OIE, 2021). It is possible to detect the subclinical form of infection caused by this bacterium in faecal samples using a PCR test (Munster et al. 2011). The test's appropriate sensitivity and speed have contributed to its widespread adoption. However, the presence of inhibitory substances in the sample can lead to false negative results and reduce diagnostic sensitivity, making the DNA extraction stage crucial for accuracy. Calcium and milk fat are known inhibitors of PCR (Schrader et al. 2012). Two proposed strategies to enhance PCR performance are enzymatic digestion and dilution of milk samples (Millar et al. 1996). The immunomagnetic method for isolating target agents has been utilized in food microbiology and veterinary laboratories. This approach has successfully detected MAP in faecal specimens, identified viral presence in individuals with AIDS (Li et al. 1996) and detected Mycobacterium tuberculosis in cerebrospinal fluid (CSF) (Mazurek et al. 1996). Furthermore, this method effectively minimizes the interference from inhibitors in PCR reactions (Khare et al. 2004; Grant et al. 2000). In prior studies, the use of immunomagnetic nanoparticles enabled the sensitive detection of MAP in colostrum, milk and faecal samples, including those from experimentally contaminated sources and faeces from cows suspected of having Johne's disease (Khosravi et al. 2021).

In a previous study, Khosravi et al. (2023) successfully employed an immunoelectric (IE) instrument to detect Escherichia coli and Staphylococcus aureus in both experimentally contaminated milk samples and clinical samples from mastitis cases. This device captures bacteria in a sample using a specific antibody on a porous polystyrene filter. The binding of bacteria to the filter, along with the adsorption of Fe nanoparticles linked to the antibody, alters the filter's electrical resistance. This change is measured with an electrical ohmmeter. With a detection time of less than 60 min, a cost‐effective price per sample, user‐friendly operation and no requirement for specialized equipment, this device represents a viable alternative to traditional bacterial detection methods in farms or dairy processing facilities. In this study, MAP bacteria were detected using both PCR and IE methods in faecal samples from dairy cows with positive and negative serum antibody titres in ELISA. The sensitivity and specificity of the IE test were compared to those of PCR and ELISA.

2. Materials and Methods

2.1. Sampling

This study was conducted on two industrial dairy farms of Esfahan province in Iran. The cows selected for testing were identified by health authorities using ELISA to detect antibodies against MAP. Rectal faecal samples were subsequently collected from 29 serum‐negative and 46 serum‐positive cows and stored at −20°C until testing. In addition, 28 faecal samples from cows, which yielded both positive and negative results in the ELISA test, were collected by the animal husbandry department of a different dairy farm and tested in a blinded manner.

2.2. ELISA Test on Serum Samples

The ELISA test was performed on serum samples using an indirect ELISA method with the ID.VET Kit. In this procedure, the serum samples were initially incubated with Mycobacterium phlei antigen to absorb cross‐reactive antibodies. Subsequently, the serum was diluted and added to the wells of the plate, along with positive and negative controls. The remaining steps were carried out in accordance with the manufacturer's instructions.

2.3. Nested PCR Test

DNA extraction from the collected faecal samples was performed using a commercial kit (CinnaGen, Iran) according to the manufacturer's instructions. The specific primer sequences for MAP (IS900) used in the PCR assays were as described in previous studies (Corti and Stephan 2002). Each PCR test included a positive control with DNA extracted from MAP and distilled water as a negative control. The PCR reactions were set up with 10 µL of Master Mix (Amplicon, Germany), 1 µL of each primer, 3 µL of extracted DNA and 5 µL of distilled water. This mixture was then placed in a thermocycler (Mastercycler Gradient, Eppendorf, Germany). The PCR cycling conditions were as follows: an initial denaturation at 94°C for 5 min, followed by 30 cycles consisting of denaturation at 94°C for 1 min, annealing at 62°C for 30 s and extension at 72°C for 30 s. A final extension phase consisted of one cycle at 72°C for 7 min. Nested PCR reactions were performed on the products of the first PCR under the same conditions with extensive care for the prevention of the PCR product pollution. The final products were electrophoresed on agarose gel containing Safe Stain (CinnaGen, Iran) alongside a 50 bp gene ladder (CinnaGen, Iran) as a reference. The gel images underwent a thorough assessment, and the band intensities were classified using a scale ranging from 0 to 3, where 0 indicates a negative result, 1 signifies weak intensity, 2 represents intermediate intensity and 3 denotes intense intensity.

2.4. Preparation of MAP‐Specific Antibodies

It is important to emphasize that all experiments involving animals were conducted in strict accordance with veterinary ethics and animal rights principles. All animals had unrestricted access to water and food during the laboratory work, were housed in well‐ventilated conditions with suitable bedding and were exposed to a 12‐h light/dark cycle.

The preparation of hyperimmune sera from rabbits and donkeys, along with the purification and characterization of specific antibodies and the synthesis of immunomagnetic beads (IMB), followed the methodology outlined by Khosravi et al. (2021). In brief, antibodies were extracted from hyperimmune sera of donkeys and rabbits using affinity chromatography. Cross‐reactive antibodies were removed through absorption with M. phlei. The purity of the antibodies was assessed using the Bradford assay and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE).

2.5. Synthesis of IMB

The Fe nanoparticle solution was prepared using the solvothermal method as described by Khosravi et al. (2021). The synthesized Fe nanoparticles were conjugated to purified antibodies against MAP using a diethylenetriaminepentaacetic acid (DTPA) linker, following the protocol outlined by Khosravi et al. (2021). In brief, DTPA powder was dissolved in a tris buffer (0.1 M, pH 9.6). The prepared Fe nanoparticles of 130 mg were suspended in the DTPA solution and incubated overnight at room temperature. After three washes with phosphate‐buffered saline (PBS), 1 mg of rabbit anti‐MAP antibody was added to the activated Fe nanoparticles, which were then shaken at room temperature for 2 h, followed by overnight incubation at 4°C. The reactive groups of the Fe nanoparticles were blocked by adding skim milk solution (4 mg/mL) and shaking for 2 h at room temperature. Subsequently, the mixture was washed three times with sterile PBS. The resulting IMB was suspended in 10 mL of a storage buffer containing sterile PBS with 1% bovine serum albumin (BSA) and 0.01% Triton X‐100. The synthesized Fe nanoparticles and IMB were further analysed using field emission scanning electron microscopy (FESEM) and Fourier‐transform infrared (FTIR) spectroscopy. The potassium bromide pellet method was employed for FTIR analysis (Perkin‐Elmer, 2400), and the spectrum was obtained through 16 scans within the absorption range of 4000–400 cm−1. The Fe nanoparticles and IMB were diluted in a 1:10 ratio, spread on aluminium foil and dehydrated by gradually increasing the concentration of ethanol from 40% to 100%. They were then fixed using a 2% glutaraldehyde solution. The prepared samples were dried in an incubator at 60°C for 72 h. FESEM (MIRA2) was utilized to determine the morphology and size of the synthesized particles.

2.6. Designing an IE Device

The components of the IE device were designed using SolidWorks, a computer‐aided design software. These components were then produced using a 3D printer at the Science and Technology centre of Khuzestan province, Iran (Figure 1).

FIGURE 1.

FIGURE 1

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The schematic representation of the immunoelectric tools includes the following elements: (a) an electric ohmmeter for assessing electrical resistance; (b) a column and reservoir filter with connection points for the ohmmeter; (c) an electric pump for circulating the sample throughout the column; (d) transfer tubes; (f) a filter for trapping bacteria and subsequently the immunomagnetic beads.

2.7. Preparation of IE Filters

The prepared polystyrene filters were activated using a DTPA linker, followed by the binding of MAP‐specific antibodies. Initially, the filters were washed with hot water and then gently placed in a container containing 10 mL of a Tris solution with a molarity of 0.1 at pH 9.6, along with 50 µg of DTPA powder. The filters were left in this mixture and shaken for 12 h before being washed again. Anti‐MAP antibodies at a concentration of 50 µg per filter were then added to each filter in a container containing 0.1 M tris at pH 8.2. Approximately 1 h later, 10 µL of a 1% glutaraldehyde solution was added to each filter. After another 12 h and a subsequent wash, 10 mL of skimmed milk solution (4 mg/mL) was added to block the filters. After 4 h, the filters were washed gently with PBS and stored in PBS buffer containing 1% BSA at 4°C until use.

To ensure that the antibodies were effectively bound to the filter and that the activated filters were adequately blocked, the filters were treated with 100 µL of a 1:3000 dilution of HRP‐conjugated anti‐rabbit antibodies (1 mg/mL) in 10 mL of PBS. Incubation was conducted at room temperature for 1 h, followed by five washes with saline phosphate buffer containing 0.05% Tween‐20. After adding 250 µL of tetramethylbenzidine (TMB) substrate to the filters, the reaction was visually assessed for up to 10 min. In the control filter, all the same steps were followed, except that 0.02 M phosphate buffer was used in place of the anti‐MAP antibodies.

2.8. Bacterial Detection in the IE System

The test samples comprised 75 faecal samples collected from a cattle farm in Esfahan province, Iran. The selected animals had previously been tested using commercial serum ELISA methods, including both MAP‐positive and MAP‐negative cows. A 2 g faecal sample was mixed with 10 mL sterilized distilled water and incubated at laboratory temperature for 10 min to sediment coarse particles. Under hygienic conditions, the filter was placed inside the columns of the device and washed three times with 200 mL of sterilized distilled water, after which the water was drained from the machine. A 200 mL of fresh PBS was introduced into the device, and the initial resistance of the filter was measured using an ohmmeter (primary resistance measurment; ER1). Next, 1 mL of the faecal sample and 250 µL IMB were added to the device. The pump was activated, allowing the sample to flow through the machine for 20 min. After this period, the device was turned off, and the solution containing PBS, IMB and faecal material was drained. The filter was then washed five times with 200 mL of PBS and completely drained. Finally, 200 mL of PBS was added to the device, and the final resistance of the filter was measured using an ohmmeter (secondary resistance measurment; ER2). A decrease in secondary electrical resistance compared to primary resistance indicates a positive sample; otherwise, the sample is considered negative. The prepared results were recorded as ER2/ER1, where ER2 represents the final electrical resistance and ER1 represents the initial electrical resistance. Results equal to or greater than one were reported as negative, whereas results less than one were reported as positive samples. All samples were tested a minimum of two times. After the test, the used filter was removed from the device, and the IE system was washed three times with distilled water containing 2% HCl. The filters used for testing the positive and negative samples were analysed using FESEM as described for imaging of IMB.

2.9. Evaluation of the IE System at the Cattle Farm Site

In this stage of evaluating the IE device, 28 cow faeces samples, which yielded both positive and negative results in the ELISA test, were collected by the animal husbandry department of a dairy cow farm in Esfahan province, Iran, and provided to the researcher in a blinded manner. The evaluation of the samples was conducted in the presence of an animal husbandry expert in the laboratory of the dairy farm, following the specified methodology.

2.10. Statistical Analysis

The results of the tests were analysed using Cochran's Q, the Spearman correlation coefficient and Cohen's kappa statistic in SPSS software (version 2022). The sensitivity and specificity of the tests were calculated using the following formulas:

  • Sensitivity = number of true positives/(number of true positives + number of false negatives)

  • Specificity = number of true negatives/(number of true negatives + number of false positives)

Plotting of the results was performed using GraphPad Prism 8 software. The results of the IE test were compared with those of the PCR and ELISA tests.

3. Results

3.1. Purification of Anti‐MAP Antibodies

Antibodies were purified from the serum of rabbits and donkeys hyperimmunized with MAP bacteria using ion exchange chromatography and affinity chromatography. The quantity and purity of the antibodies were measured using the Bradford protein assay and SDS–PAGE electrophoresis. The SDS–PAGE results demonstrated a single band on the gel (Figure 2), indicating successful purification of immunoglobulin G (IgG) from the serum of both donkey and rabbit subjects. The final concentrations of the antibody fractions of rabbits and donkey, which exhibited adequate purity and concentration, were adjusted to 300 µg/mL. The conjugation of purified rabbit antibodies to the Fe nanoparticles was successfully confirmed through FTIR results (Figure 3).

FIGURE 2.

FIGURE 2

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Evaluation of Mycobacterium avium subspecies paratuberculosis IgG antibody purity using polyacrylamide gel electrophoresis.

FIGURE 3.

FIGURE 3

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Fourier‐transform infrared (FTIR) spectroscopy analysis of the prepared immunomagnetic beads. The redshift of the spectral peaks from 675 to 728 (cm−1), respectively, along with the emergence of new peaks 949 and 1014 (cm−1), indicates C–O and C–H bending vibration and incorporation of antibodies into the Fe nanoparticles. (Blue spectra) Fe nanoparticles, (red spectra) antibodies and (green spectra) conjugated antibodies to Fe nanoparticles.

3.2. Optimization of Antibody Linking to IE Filters

The binding of MAP‐specific donkey antibodies to a polystyrene filter was evaluated under various conditions. The optimal binding conditions involved initially activating the filter with 100 mg of DTPA in a 0.1 M Tris buffer (pH 8.2), followed by overnight incubation at 4°C. After washing the filter with Tris buffer, 200 µg of antibodies were added to the activated filter, which was then shaken at room temperature for 2 h. To stabilize the antibody binding, a 1% glutaraldehyde solution was introduced to the mixture. To cover the unoccupied areas of the filter, skimmed milk was added in a phosphate saline buffer (4 mg/mL), and the mixture was incubated on the shaker for an additional 2 h before washing the filter. The prepared filters were then placed in a phosphate saline buffer containing 1% serum albumin (pH 7.2) and stored at 4°C.

3.3. Detection of MAP Contamination in Cow Faecal Samples

Faecal samples from both healthy and MAP‐infected cows were analysed using PCR and IE methods. The results are presented in Tables 1, 2, 3. The analysis revealed a significant decrease in the electrical resistance of the device's filter in certain samples, characterized by a reduction in the ratio of secondary resistance to baseline resistance; these were subsequently classified as positive (Table 2). The capture of MAP bacteria on IE filters was successfully observed in FESEM images (Figure 4).

TABLE 1.

Detection of cows with Johne's disease using polymerase chain reaction (PCR) and serum enzyme‐linked immunosorbent assay (ELISA) methods.

Assay PCR+ PCR− Total
ELISA+ 40 6 46
ELISA− 13 16 29
Total 53 22 75
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Note: Sensitivity of ELISA relative to PCR = (40/53) × 100 = 75%. Specificity of ELISA relative to PCR = (16/22) × 100 = 72%. Agreement of ELISA and PCR = (56/75) × 100 = 74%. The kappa statistic is equal to 0.44.

TABLE 2.

Detection of cows with Johne's disease by polymerase chain reaction (PCR) and immunoelectric methods.

Assay PCR+ PCR− Total
Immunoelectric + 51 1 52
Immunoelectric − 2 21 23
Total 53 22 75
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Note: Sensitivity of immunoelectric relative to PCR = (51/53) × 100 = 96%. Specificity of immunoelectric relative to PCR = (21/22) × 100 = 95%. Agreement of immunoelectric and PCR = (72/75) × 100 = 96%. The kappa statistic is equal to 0.91.

TABLE 3.

Detection of cows with Johne's disease by serum enzyme‐linked immunosorbent assay (ELISA) and immunoelectric methods.

Assay ELISA+ ELISA− Total
Immunoelectric + 38 14 52
Immunoelectric − 8 15 23
Total 46 29 75
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Note: Sensitivity of immunoelectric relative to ELISA = (38/46) × 100 = 82.6%. Specificity of immunoelectric relative to ELISA = (15/29) × 100 = 51.7%. Agreement of immunoelectric and ELISA = (53/75) × 100 = 70.6%. The kappa statistic is equal to 0.36.

FIGURE 4.

FIGURE 4

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Field emission scanning electron microscopy (FESEM) images of the prepared (a) Fe nanoparticles and (b) immunomagnetic beads. Panels (c), (e) and (f) demonstrate the capture of MAP bacteria on immunoelectric filters, whereas panels (d), (g) and (h) illustrate the capture of MAP bacteria in conjunction with immunomagnetic beads (indicated by red arrows) on immunoelectric filters.

These findings showed a strong correlation between the results obtained from the IE system and the PCR method. Using Cochran's test, it was determined that there was no significant difference between the PCR and IE diagnostic methods (p = 0.14).

The correlation coefficient between the degree of contamination of the samples analysed using the PCR method and the reduction of electrical resistance measured by the IE ohmmeter is 0.66, indicating a strong inverse relationship.

When comparing the sensitivity and specificity of the ELISA method to PCR, the values were 75% and 72%, respectively. Notably, the IE method demonstrated higher sensitivity and specificity, with values of 96% and 95%, respectively, when compared to PCR. Furthermore, the agreement between PCR and IE tests is 96%, whereas the agreement of the ELISA method with PCR is only 74% (Figure 5a, Tables 1 and 2 and Supplementary File S1).

FIGURE 5.

FIGURE 5

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Evaluation of changes in electrical resistance (mean ± standard error) in an immunoelectric system for assessing contamination of cattle faecal samples (n = 75) with Mycobacterium avium subspecies paratuberculosis. (a) This analysis employs immunoelectric methods, PCR and ELISA. (b) This analysis employs immunoelectric and ELISA at a laboratory cattle farm site (n = 28). Single asterisk (*) denotes a significance level of <0.01, two asterisks (**) indicate a significance level of <0.05 and three asterisks (***) represent a significance level of <0.001. ELISA, enzyme‐linked immunosorbent assay; PCR, polymerase chain reaction.

3.4. Results of the Evaluation of the IE System at a Cattle Farm Site

In this evaluation, 28 samples were tested using the IE method. The results showed an equal distribution, with 14 samples testing positive and 14 testing negative. The serum ELISA test results indicated 12 positive samples, of which 11 also tested positive with the IE method. Among the 17 samples that were negative in the ELISA test, 14 were also negative in the IE test (see Figure 5b and Supplementary File 1).

4. Discussion

Johne's disease is a notable infectious disease impacting ruminants globally, leading to substantial economic losses within the dairy cattle industry. Various factors complicate efforts to control this disease, including the prolonged incubation period of the causative bacterium, the challenges in diagnosing subclinical cases and the limited availability of accurate, rapid and cost‐effective diagnostic tests. Furthermore, there is a lack of comprehensive understanding of the different strains of MAP and the complexities involved in interpreting diagnostic results (Sohal et al. 2007). The newly developed IE method, like other diagnostic tests, has notable advantages and limitations. Currently, direct diagnostic methods are limited to bacterial culture, PCR, real‐time PCR and Ziehl–Neelsen staining. Unlike prolonged bacterial culture, the IE method allows for separation and detection steps without the disadvantages associated with culture, such as contamination; however, unlike bacterial culture, the method could not differentiate live and dead cells. Additionally, this method does not require initial extraction steps and utilizes inexpensive and straightforward components, making it more accessible than PCR equipment. The current research compares the sensitivity and specificity of the IE method to PCR for the detection of the MAP in cow faecal samples. The immunologic reactions in the IE tool are based on the capture of antigens using an activated filter with a specific antibody. The binding of the Fe nanoparticles to the captured bacteria is mediated by secondary specific antibodies.

On the basis of the findings of the current study and prior research by Khosravi et al. (2021), the use of MAP‐specific polyclonal antibodies after removing antibodies that cross‐react with other mycobacteria through proximity to M. phlei results in detection with sufficient sensitivity and specificity in sandwich assays. The successful application of polyclonal antibodies in this study aligns with the findings of Karuppusamy et al. (2021), who effectively used polyclonal antibodies to target the cell envelope. They isolated MAP using IMB prepared by polyclonal antibody and successfully detected it via PCR with a detection limit of 100 cells.

The designed IE tool tests one sample per run, which limits the device's usage for screening on the farm. By utilizing a larger volume of tested samples, the IE method offers the potential for greater sensitivity than known direct bacterial detection methods. There are no known or reported IE methods for bacterial detection to compare with the currently developed method; however, we have found reports regarding the limited efficiency of other direct detection methods. As an example, Clark et al. (2008) employed the PCR method to directly assess cow faecal sample contamination with MAP. The sensitivity and specificity of this method were found to be 70.2% and 85.3%, respectively. Moreover, in a comparative study conducted by Garg et al. (2015) involving 62 cattle, both PCR and antibody detection by ELISA methods were utilized. The results indicated that 27 cows have a negative result with both methods, and 25 cows have positive result with both ELISA and PCR, whereas 5 cows were positive for ELISA only and another 5 were positive for PCR only. The discrepancies in results of different diagnostic methods may stem from variations in the types of methods used, differences in contamination levels among the livestock evaluated and the size of the animal populations studied. Additionally, the observed differences in seroprevalence studies can be attributed to factors such as climatic variations among countries, herd management practices, herd age and the number of susceptible animals within each herd and region. Other contributing factors include variations in sampling methods, the volume of samples needed for testing and the quality of the employed test elements. The primary challenge in effectively applying PCR‐based diagnostic methods is the insufficient quality of extracted DNA. This issue stems from the robustness of the mycobacterial cell wall and the presence of various PCR inhibitory compounds. Therefore, improving DNA extraction techniques is essential, as proper sample preparation is critical for obtaining high‐quality, high‐purity DNA (Sting et al. 2014). This magnet‐based separation method has shown effectiveness in the detection and isolation of MAP. Additionally, the combined use of immunomagnetic nanoparticles and IS900 PCR significantly reduces the time needed for MAP isolation and detection (Grant et al. 2000). In the present study, the continuous flow of the sample in an appropriate buffer enhances the binding of the immunomagnetic nanoparticles to the bacteria and the IE filter, thereby facilitating the effective isolation and diagnosis of the bacteria in a single step. The potential for using mixed samples to track MAP in faecal samples through faecal culture and real‐time PCR was evaluated by Wichert et al. (2021). They suggest that in areas with high contamination levels, combining up to 10 samples has a minimal impact on the sensitivity of diagnostic methods compared to mixing 5 samples. The need for specialized equipment and the costs associated with bacterial culture and PCR diagnostic methods have led to the evaluation of mixed sample testing and its outcomes. Nonetheless, the current method employed in this study is cost‐effective and delivers results in less than 60 minutes, allowing for the analysis of all samples without the need for mixing. Nevertheless, the application of pooled samples may offer an opportunity to utilize IE methods for screening of farms. The introduction of new methods and technologies has the potential to improve diagnostic procedures. The IE method, in particular, addresses some limitations of the previously mentioned techniques concerning testing conditions, time and cost.

In most laboratory methods, 200 mg to 1 g of bovine faecal samples are mixed with water (Khare et al. 2004; Salgado et al. 2013). To assess the sensitivity and diagnostic efficacy of various techniques, experimentally contaminated samples are also utilized. A notable aspect of the present study is the evaluation of clinical specimens, distinguishing it from other research efforts. Herrold's egg yolk agar medium demonstrates 30%–50% sensitivity and 100% specificity; however, the method requires 2–3 months, making it impractical for timely clinical diagnosis. The ELISA is employed for indirectly detecting infection of the animals by MAP bacteria, but its sensitivity and specificity depend on the progression of the disease. Therefore, positive samples identified by the ELISA method should be re‐evaluated using culture or PCR tests (Clark et al. 2008).

In a study involving 147 cows confirmed to be infected with MAP, 80 cows (55%) tested positive using a combined method of immunomagnetic isolation and PCR. In contrast, only 2 cows (1.4%) tested positive via the milk culture method, 15 cows (10%) via faecal culture and 20 cows (14%) via ELISA (Gilardoni et al. 2016). These findings suggest that the stage of the disease and the mode of infection significantly influence the sensitivity of the diagnostic methods. The IE method can be employed to detect MAP in the faeces of livestock, which is crucial for controlling and preventing disease. It appears that bacterial detection methods in faecal samples with techniques such as PCR and culture can yield more reliable results than serological methods like ELISA. In the current study, we found agreement value of the ELISA results with the PCR and IE results to be 74% and 70.6%, respectively.

A comparison of the Ziehl–Neelsen method and PCR demonstrated a moderate agreement rate (0.571) in detecting MAP in faecal samples from small ruminants (Dixit et al. 2023). In the mentioned study, 36 samples that tested positive with the Ziehl–Neelsen method yielded negative results with the PCR test, whereas 7 samples that were negative by Ziehl–Neelsen turned out to be positive in the PCR test. In our study, the sensitivity and specificity of the IE method were found to be equal to 96% and 95%, respectively, when compared to PCR. However, two samples that were negative in the PCR test tested positive when assessed using both IE and Ziehl–Neelsen methods. This inconsistency in results may be attributed to minor contamination and a lack of uniformity in sampling for DNA extraction. In summary, although various diagnostic techniques exist for detection of Johne's disease, the choice of method should consider the balance among sensitivity, cost and the urgency of obtaining results, particularly in field settings. Continued advancements in diagnostic methodologies will be crucial for improving the management and control of this challenging disease.

5. Conclusion

The IE method demonstrates promising sensitivity and specificity for the detection of MAP in feces, with values of 96% and 95%, respectively. The kappa statistic was calculated to be 0.91. This method has emerged as a more cost‐effective alternative to the PCR approach, delivering results with comparable sensitivity in a shorter timeframe. Additionally, the IE method can be utilized in farm laboratories. However, the current design of the IE device tests only one sample per run, which limits its applicability for screening on farms. Therefore, further development is needed to optimize its use in the agricultural industry.

Author Contributions

Mohammad Khosravi: investigation, writing original draft, review and editing, methodology, resources, project administration, funding acquisition, conceptualization. MohammadRahim Haji Hajikolaei: project administration, formal analysis, conceptualization. Mohammad Nouri: review and editing, formal analysis, conceptualization. Shayan Kalanter: investigation, writing original draft, formal analysis.

Ethics Statement

All tests on animals were carried out in accordance with animal protection laws and guidelines of research ethics committee of Shahid Chamran University of Ahvaz and approved with the verification code of IR.SCU.REC.1402.018.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/vms3.70346.

Supporting information

Supporting information

VMS3-11-e70346-s001.xlsx (16.4KB, xlsx)

Acknowledgements

The authors express their gratitude to the management and staff of FOKA and Fazil Industrial Cattle Farm in Isfahan for their collaboration in furnishing clinical samples.

Funding: This study was financially supported by the Shahid Chamran University of Ahvaz, Ahvaz, Iran grant.

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 information

VMS3-11-e70346-s001.xlsx (16.4KB, xlsx)

Data Availability Statement

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

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