Abstract
Background
This study has investigated designing, producing, and evaluating a newly developed immunoelectric device for the identification of the Staphylococcus aureus and Escherichia coli in experimentally or clinically polluted milk samples.
Methods
The design of the immunoelectric tool parts was carried out and produced by using a 3D printer. The animals were immunized against S. aureus and E. coli; purification of the specific IgG from hyperimmune serum was carried out by ion exchange and affinity chromatography methods. Coupling of the specific rabbit antibodies to the polystyrene filters was performed by DTPA linker. The purified rat antibodies were coupled to Fe nanoparticles and used as detector elements. The experimentally polluted milk and PBS samples were prepared. After optimization, the minimum traceable number of the bacteria was determined using immunoelectric tool. Also, the cow's milk samples with clinical mastitis were tested for bacterial detection by bacterial culture and immunoelectric methods.
Results
Coupling of the antibody to the filters and capturing of the target bacteria to the filters were successfully confirmed using enzyme immune assay and electron microscopy. The detection limit of the developed immunoelectric tool was equal to 15 cells/mL in PBS or milk samples for 30 min. Also, in agreement with bacterial cell culture, the clinically infected milk samples by S. aureus and E. coli were successfully detected using the immunoelectric device.
Conclusions
The developed immunoelectric device could be utilized for rapid and cost–benefit detection of the bacterial organism.
Keywords: antibody, bacteria, Immunoelectric, mastitis, milk
Schematic trapping of the target bacteria and immunomagnetic beads on polystyrene filter. The immunoelectric instrument rotates the test sample in the embedded tracking column to properly trap the target bacteria on a polystyrene filter; the subsequent binding of the specific immunomagnetic beads to the target bacteria provides a suitable change of the electric resistance which could be detected by an ohmmeter.
1. INTRODUCTION
Inflammation of the mammary gland parenchyma leads to a decrease in the quality and level of milk production in dairy cows. 1 The suitability of a diagnostic method could be recognized by factors such as cost, specificity, sensitivity, time, and automation to check the large number of milk samples. 2 A rapid test is required in clinical cases to diagnose the disease agent before that clinician apply the treatment methods. Any delay in correct diagnosis of the agents can lead to elevation of the disease period as the use of drugs without therapeutic effects. 3 The milk culture testing and somatic cell counting are commonly used for the detection of mastitis agents. 1 , 4 Culture of milk samples, while having a high cost and time‐wasting, is a reliable test to diagnose subclinical mastitis. 5 Polymerase chain reaction (PCR) and bacterial culture are usual and reliable tests for tracing of the pathogenic bacteria. The concurrent utilization of bacterial culture and PCR has higher sensitivity, so these tests are used together to achieve greater sensitivity. However, due to its time‐consuming and cost, this approach delays effective treatments. 2 Despite the several commercial enzyme‐linked immunosorbent assay (ELISA) kits for diagnosing mastitis, the test did not detect the small quantities of antigens. 6 Other disadvantages of using current diagnostic methods include the impossibility of testing in most animal farms and veterinary clinics; the usual tests always have high costs and need a laboratory expert to perform the diagnostic assay. 2 Field screening tests such as somatic cell count or California mastitis tests usually used for detecting subclinical mastitis cases; however, they did not identify the cause of the disease. 7 The electrical conductivity of milk resulted in false‐positive outcomes; it is not a reliable method for an accurate and definitive diagnosis of mastitis. 8
Biosensors are used for rapid and low‐cost diagnosis of pathogens. Conventional biosensors use a signal‐generating transducer that may be electrochemical, optical, or mechanical. The electrochemical biosensors are desirable due to their low cost. 9 These assays have a lower detection limit for early‐stage monitoring, reduce the detection time, and increase the detection accuracy of the target in the complex unprocessed matrix and identify the desired factor in multi‐microbial samples. 10
The E. coli bacteria are pathogenic in humans, animals, and plants. Staphylococcus aureus bacteria cause various kinds of fatal infections. According to this requirement, many approaches were developed to reduce the time of detection, cost of the test, or trained personnel. 11 Infection dosages of some pathogenic microorganisms are less than 4 cells/100 mL of water or 1 cell/25 gr of food samples; the current detection approach could not fulfill these requirements. 12 Today, the developed electrochemical biosensors are based on direct detection of the bacteria or indirect detection of the bacterial metabolites in different samples; these strategies provided promising results. 10 The dairy industry and domestic livestock breeding demand rapid and accurate diagnostic methods. Enlargement of cattle farms, milk production, and applying milking machines need hygienic conditions for cattle breeders. This study developed a new method to track S. aureus and E. coli in milk samples using an immunoelectric tool.
2. MATERIALS AND METHODS
2.1. Preparation of the bacteria
The bacteria were achieved from bacterial archives of the Microbiology Department, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz. The Staphylococcus aureus and Escherichia coli bacteria were cultured on blood agar or MacConkey agar medium, respectively. The cultured plates were incubated at 37°C for 48 h. Isolated clones were identified using gram stain and biochemical tests including coagulase, DNase, sugar fermentation, catalase and oxidase, TSI reaction, IMViC, and urease production; the positive colonies were counted manually. The net bacterial samples were placed in PBS containing 0.5% formaldehyde for 24 h at laboratory temperature. After that, formaldehyde‐treated bacteria were cultured in blood agar and MacConkey medium after three times centrifugation and washing with PBS. After incubation for 48 h at 37°C, the negative results of bacterial growth indicated a successful inactivation of the bacteria.
2.2. Preparation and evaluation of the hyperimmune serum
The animals, including four rabbits and 12 rats, were kept at 22°C in the animal house at the veterinary faculty of the Shahid Chamran University of Ahvaz, Iran. They have free access to water and food. Animals were placed in separate cages for immunization with S. aureus or E. coli bacteria. The inactivated bacteria 0.5 mL of 6 × 108 cells/mL were suspended in an equal volume of Freund complete adjuvant. After gently mixing, antigens were injected subcutaneously and intramuscularly 1 mL per rabbit and 250 μL for each rat. The immune responses against mentioned bacteria were boosted by three booster injections of 3 × 108 cells/mL of each bacteria mixed with an equal volume of the incomplete Freund adjuvant at 2‐week intervals.
The blood samples of 1 mL were taken from the ear marginal vein of rabbits or the lateral tail vein of the rats to evaluate the humoral immune responses. The blood samples were stored at room temperature for 1 h and 2 h at 4°C temperature and then centrifuged at 1000 g for 10 minutes. The microagglutination test (MAT) was used to evaluate the sera antibody titers as usual. After ensuring the suitable antibody titer, the animals were anesthetized using a combination of xylazine‐ketamine 5:50 mg/Kg, and blood samples were collected from the hearts of the animals. The hyperimmune serum was separated as before.
2.3. IgG antibodies isolation
Precipitation of IgG antibodies was performed using ion exchange chromatography according to Westwood and Hay (2012). 13 The specific antibodies against S. aureus and E. coli bacteria were purified using an affinity chromatography column. Briefly, formalin‐killed bacteria were sonicated, and 5 mg of the prepared bacterial antigens coupled to activate Sepharose 4B by a cyanogen bromide linker. The extracted total IgG was incubated in the activated column for 1 h; the column was gently washed with PBS to remove the other antibodies. The specific antibodies bound to the column were released using a glycine buffer (pH 2.5) and immediately neutralized using Tris buffer (pH 9.5). The level, purity, and activity of the antibodies were defined using Bradford, sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), and ELISA methods, respectively. Sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel was prepared as 11% separating gel and 4% polyacrylamide stacking gel according to a common protocol.
2.4. Designing of the immunoelectric device
Different parts of the immunoelectric device including filters for trapping the target antigen (Figure S1), a column for holding the filter with resistance measurement connections, caps, and holders of the columns were designed using the solid work computer‐aided design (CAD) software. Designed parts were produced using a 3D printer with High Impact Polystyrene (HIPS) by Ideh Prdaz Ebtekar Avizeh Co, Ahwaz, Iran. These parts are shown in Figure 1.
FIGURE 1.
The designed parts of the immunoelectric device. (A1, A2) Trapping filters which have two orifices for linking to the connector of the ohmmeter electrodes. (B1–B3) Caps of the tracking, wash buffer, and reaction buffer columns. (C1, C2) Holders of the columns. (D1–D4) Various designed parts for tracking column which include two fragments for the holding of the filter and outlet for the connector of the ohmmeter electrodes; (E) glass tubes form the columns (F) immunoelectric device.
2.5. Coupling of the trapping antibody to the filter
To activate the produced filters, the purified antibodies were coupled to them using diethylenetriamine pentaacetate (DTPA) linker; first, the filter was washed with warm water to remove the remained glue. After that, it was placed in 0.1 M Tris solution pH 9.6 which contains 100 mg DTPA. The treated filters were incubated in this solution for 12 h at room temperature and then rinsed carefully. The purified specific antibacterial rabbit antibodies against S. aureus, or E. coli were added in concentrations of 10 μg/mL in a flask containing 10 mL of PBS, pH 8.2; the DTPA‐activated filter was incubated in this PBS‐containing antibody for 1 h at room temperature; after that, 1 mL of 1% glutaraldehyde solution was added to the reaction. After 12 h of incubation at 4°C, the filter was carefully rinsed again. Skimmed milk solution of 4% was added into the flask and shacked for 2 h at room temperature to block the unreacted chemical groups of the filter. Finally, the activated filter was rinsed again with PBS and stored in a PBS buffer containing BSA 1%. The control filters were prepared as mentioned before, except that instead of the antibody, 0.02 M phosphate buffer without antibody was added to the flask that contain the DTPA‐activated filter. The antibody coupling to the filter and proper blocking of the unreacted group of the filter was evaluated using an enzyme immune assay. The horseradish peroxidase‐conjugated anti‐rabbit antibody 1 mL of 1/30.0000 dilution was added to the activated and control filters. After 1‐h incubation at room temperature, five‐time washing was performed with PBS containing 0.05% Tween 20. After that, 250 μL of 3,3′,5,5′‐Tetramethylbenzidine (TMB) substrate was added to the washed filter, and the reaction was evaluated visually until 10 min.
2.6. Coupling of the detector antibody to the Fe nanoparticles
According to Khosravi et al. (2021), 14 Fe nanoparticles were produced by the heat solvent method. Then, DTPA 200 mg was dissolved in 1 mL of DMSO; the volume of the mixture was adjusted to 5 mL using 0.1 M Tris–HCL pH 7.2; 10 μL of Triton X 100 was added to the reaction, and the pH of this mixture was adjusted to 9.6. Then, 1 mL of Fe nanoparticles, with a concentration equal to an optical density of 0.312 at a wavelength of 600 nm, was added to the mixture. After 2 h of shaking at room temperature, the mixture was incubated at 4°C overnight. The Fe nanoparticles were then collected using a magnet and washed three times with sterilized PBS pH 7.2. After washing, the mixture was adjusted to the original volume using Tris–HCL 0.1 M, pH 8.2. The specific rat antibodies against S. aureus or E. coli 1 mL, 300 μg/mL, were added separately to the activated Fe nanoparticles. The resulting mixture was then shaken gently for 2 h and then kept at 4°C for 24 h. The produced immunomagnetic beads were blocked with 4 mg/mL of skimmed milk for 2 h. The Fe nanoparticles were then collected with a magnet and washed three times with sterilized PBS. The resulting immunomagnetic nanoparticles were supposed in PBS containing 1% BSA in a volume equal to baseline, and the resulting mixture was stored at 4°C.
2.7. Evaluation of the bacterial trapping by electron microscopy
Staphylococcus aureus and E. coli bacterial trapping into the activated filters were evaluated experimentally and naturally polluted PBS and milk samples. The treatment procedure was the same as in the previous section. The preparation steps of the filters for SEM microscopy included dewatering at increasing concentrations of ethanol (10%–100%) and fixing with glutaraldehyde. Then, sections with a diameter of 0.5 cm and height of 2–3 mm were removed randomly from the filters and inspected by scanning electron microscopy.
2.8. Detection of the trapping bacteria onto the immunoelectric filter
The designed immunoelectric device consists of 3 columns, including 1—tracking column, which contains a part for trapping filter and electrodes of the electric ohmmeter; 2—columns containing PBS pH 7.2 as a test buffer and 3—columns containing PBS pH 7.2 containing 0.05% tween 20 as a washing buffer (Figure S2). The filter was placed in the tracking column and washed three times. The electric resistance and current flow of the filter were measured using an ohmmeter at time 0. The test samples were inserted into the tracking column using a syringe in a volume of 1 mL. After adding test buffer, sample was rotated through the tracking column continuously for 20 min using an electric pump. At the end of this period, the column was washed with a washing buffer three times. The detection sensitivity was increased by the addition of 100 μL of the specific immunomagnetic beads injected into the tracking column at the same time as the sample (Figure S3). The filter was washed with a washing buffer, and the electric resistance and current passing through the filter at the end time of the test were measured using an electric ohmmeter. The ratio of the electrical resistance was calculated as RET0/RET20; the RET0 and RET20 were electrical resistance at time 0 and after 20 min at the end of the experiment, respectively. The calculated value greater than 1 showed a reduction in the electric resistance.
2.9. Evaluation the optimum time for bacterial tapping to the filter
Test was performed by injection 1 mL of E. coli with a concentration equal to 3 × 105 into the tracking column containing the activated specific filter. The electric resistance and current change were detected at intervals of 5–25 min as described above.
2.10. Detection of S. aureus and E. coli contaminated samples
The immunoelectric device was evaluated for S. aureus and E. coli detection at concentrations equal to 6 × 108–30 cells/mL. Also, false results and cross‐reactions were tested on the control filters, it includes the not‐activated filters and activated filters with the other antibody. The specific filter of E. coli and S. aureus were evaluated for the occurrence of the crossing reactions. The specific E. coli filter was placed in the tracking column and then experimentally polluted PBS with S. aureus 1 mL injected into the column and vice versa. After testing, the results were recorded.
2.11. Detection of the clinical mastitis milk samples
The milk samples were collected from 50 cows with clinical mastitis suspected to E. coli or S. aureus infection. All the collected milk samples were cultured in nutrient agar medium. The plates were incubated for 24 h at a 37°C incubator. Isolated clones were identified using gram stain and biochemical tests as mentioned before. The initial electric resistance of the specific filters was defined in the range of 200 m–20 k. Subsequently, the cow milk samples 1 mL in combination with the specific immunomagnetic beads were injected into the tracking column. The following procedure included 20 min sample rotation into the tracking column and five washing steps with the washing buffer. The electric resistance of the filter was measured at the end of the test.
3. RESULTS
3.1. Evaluation of the antibody purity
Purification of the rat and rabbit's IgG against S. aureus and E. coli was performed using Ion exchange and affinity chromatography. The SDS‐PAGE results showed a single band in each lane of the gel, which indicates the successful separation of the IgG (Figure S4). The released fractions that contain antibodies were mixed, and the final concentrations were adjusted to 300 μg/mL.
3.2. Optimization of the antibody coupling to immunoelectric filter
As shown in Figure 2, unlike the control filters, the activated filters reacted with HRP‐conjugated anti‐rabbit antibodies. In the control filter, the same steps were performed, except that in activation step of the filters, 0.02 M phosphate buffer without antibody was added to the filter.
FIGURE 2.
Evaluation of the antibody coupling to the immunoelectric filters. The filters were activated using a DTPA linker and coupled to specific rabbit antibodies against (A) Escherichia coli, (B) Staphylococcus aureus bacteria, or (C) without antibodies. Antibodies binding to each filter were evaluated by adding HRP‐conjugated anti‐rabbit antibody, then washed properly using PBS, and then, TMB substrate was added to each reaction.
In addition, the trapping of S. aureus and E. coli bacteria to the treated filter were evaluated in each sample using electron microscopy. As shown in Figure 3, unlike the control samples, bacteria were observed in the activated filters. As seen, bacterial entrapment was captured at the edges of the filters.
FIGURE 3.
Scanning electron microscope (SEM) image of the immunoelectric filters. (A, B) filters that reacted with unpolluted samples, (C, D) filters that reacted with clinical milk samples suspected to Escherichia coli infection, (E, F) clinical milk samples suspected to Staphylococcus aureus infection. Sections prepared from the tested filters and evaluated using SEM. The trapped bacteria are marked by an arrow.
3.3. Optimization of the reaction time for trapping bacteria onto the filter
Escherichia coli 3 × 105 cell/mL were inserted into the tracking column which contains the activated filter; change in electric resistance of the treated filter was measured at intervals of 5–25 min; as seen in Figure 4, the best results were defined after 20 min rotation of the polluted sample in tracking column.
FIGURE 4.
Evaluation of the appropriate time for Escherichia coli coupling to the immunoelectric filter. Changes in electric resistance of the filter were recorded at intervals of 5–25 min.
3.4. Detection of the target bacteria using immunoelectric
The detection limit of the immunoelectric tool was evaluated for detection of the S. aureus and E. coli. According to the optimization conditions, detection of the S. aureus or E. coli less than, respectively, 6 × 106 or 3 × 103 cell/mL in experimentally contaminated PBS samples, and in all of the milk samples were performed by using 100 μL immunomagnetic beads in each reaction (Tables S1–S4). As revealed in Figures 5 and 6, often there was a noticeable reduction in the electrical resistance of the immunoelectric filter depending on the bacterial concentration. Maximum decrease in electric resistance was recorded in the 20 m. Also, no significant change was observed in the electrical current of the immunoelectric filter. Also, there is a noticeable decrease in the electrical resistance of the immunoelectric filter depending on the bacterial concentration. The minimum detection limit of E. coli and S. aureus was equal to 15 cells/mL of milk samples (Tables S5 and S6).
FIGURE 5.
Detection of the Staphylococcus aureus by using immunoelectric device. The low detection limit was defined for tracking S. aureus in experimentally polluted (A) PBS or (B) milk samples.
FIGURE 6.
Detection of various concentrations of Escherichia coli using immunoelectric device. The low detection limit was defined for tracking Escherichia coli using (A) PBS or (B) milk samples.
3.5. Evaluation of false reaction of the immunoelectric device
The immunoelectric filters, activated by specific antibodies against E. coli or S. aureus, were used to evaluate the crossing reactions with other bacteria in PBS or milk samples. Presence of other bacteria, like a milk sample without bacteria, did not have a significant effect on electric resistance; however, a slight decrease was recorded in some samples (Tables S7–S11). Also, the immunoelectric filters were evaluated by using the control filters. The experimentally contaminated PBS or milk samples by S. aureus or E. coli were inserted into the columns. The results show a slight elevation in the electric resistance of the immunoelectric filter after continuous washing in the tracking column (Figure 7).
FIGURE 7.
Evaluation of the false reaction of the immunoelectric filters. Specific Staphylococcus aureus filters were treated with experimentally contaminated (A) PBS or (B) milk samples by Escherichia coli. After that, the filters were used for S. aureus detection. Specific E. coli filters were treated with experimentally contaminated (C) PBS or (D) milk samples by S. aureus. After that, the filters were used for detection of the E. coli. (E) The electric resistance of the antibody‐free filter was defined before and after encountering E. coli and S. aureus bacteria.
3.6. Evaluation of milk samples of clinical cases of mastitis
The cow's milk samples suspected to E. coli or S. aureus infection were evaluated using the immunoelectric device. Out of all, a significant decrease in electrical resistance was observed in the positive samples (as sample number 3 in Figure 8A). The milk samples were cultured in nutrient agar; after biochemical identification, results showed complete agreement with immunoelectric device. Also, according to the mentioned method, milk samples suspected mastitis with S. aureus were evaluated by immunoelectric device. A significant decrease in electrical resistance was defined in the positive samples (as sample number 1 in Figure 8B). The milk samples were cultured in blood agar; after biochemical identification, the results showed complete agreement with immunoelectric device.
FIGURE 8.
Evaluation of milk samples of clinical cases of mastitis. The immunoelectric device was evaluated for tracking of (A) Escherichia coli or (B) Staphylococcus. aureus infection in milk samples of clinical cases of mastitis.
4. DISCUSSION
The results showed the successful detection of at least 15 cells/mL of S. aureus and E. coli in less than 30 min. The device detected the experimentally and clinically polluted milk samples. These results were confirmed using bacterial culture and biochemical tests. The milk samples of cases with clinical mastitis that had negative results for the presence of S. aureus and E. coli, contained other bacteria, including Streptococcus, Corynebacterium, and Klebsiella strains. In recent years, several new methods have been used to detect pathogens in various samples, including milk. Suaifan et al. (2017) 15 designed a biosensor that could be detected 100 CFU/mL of S. aureus in 1 min. The used sensor was based on the proteolysis activity of S. aureus protease enzymes, which act on a specific peptide substrate placed between two magnetic nanoelectrodes; decomposition of the peptide by magnetic nano‐electrode leads to discoloration in the substrate and detection of the bacteria. Zheng et al. (2019), 16 used a micro‐based biosensor reinforced with gold nanoparticles for quantitative detection of foodborne bacteria including E. coli H7: O157, S. aureus, L. monocytogenes, and Shigella without using bacterial culture and PCR. The detection limit was reported to be 1–9 cell/mL. The specific gene sequence analysis of the target bacteria by using the developed device was utilized for defining the bacterial concentration. In another study, Etayash et al. (2016) 17 detected E. coli and L. monocytogenes that passed through a microcanal with the aim of tracking the drug‐resistant bacteria; the inner surfaces of the canal were equipped with physical and chemical receptors of the target bacteria. The trapped bacteria were stimulated using infrared radiation; it caused a deviation corresponding to the infrared wave's absorption by the target bacteria. The nanomechanical spectrum of the infrared radiation was used to identify the concentration of the target bacteria. The minimum detectable concentration for the device was one cell per microliter of sample.
The photochemical stabilization of antibodies to a gold electrode was used for the detection of E. coli in water samples 18 ; the reported detection limit was 30 CFU/mL in 1 h. Also, Wong and Alocilja (2015) 19 developed a combined method based on immunomagnetic separation and electrochemical measurement of the Au‐antibody conjugate. This approach had a detection limit of 10 CFU/mL. Mannoor et al. (2010) 20 acclaimed that a portable electronic pathogen detector consists of a chip coated with magainin‐I, and electronic read‐out monitoring electrodes can detect E. coli. Reich et al. (2017), 21 used nucleotide‐based aptamers for detection of the S. aureus; the designed aptamer targets the bacterial protein A. This biosensor detected 10 CFU/mL of S. aureus in 10 min. Pandey et al. (2017) 22 detected E. coli H7: O157 in food samples by using a biosensitive sensor. H7‐specific antibodies were conjugated to a microelectrodes for specific trapping of the target pathogen. The detection limit of this device was reported to be 10–100 cell/mL in 30 min. Wilson et al. (2019) 23 developed an approach based on capturing the bacteria by using Fe nanoparticles conjugated to melittin peptide and impedance detection of the particles. Despite the method's sensitivity, it needs more investigation to improve the test specificity. The impedance spectroscopic biosensor is based on the fixation of the target bacteria on a planar indium‐tin oxide via specific antibodies; after that, an enzyme conjugated to an antibody interacted with the bacteria. The produced insoluble product on the surface of the electrode creates a detection signal. Currently, label‐free electrochemical impedance biosensors are more in interest for development. The detection limit of 106 CFU/mL was reported for this approach (Yang and Bashir, 2008). As can be interpreted, our immunoelectric tool is based on a different strategy for capturing and detecting antigens.
5. CONCLUSION
The immunoelectric instrument rotates the test sample in the embedded tracking column; this issue helps trap the target bacteria in the polystyrene filter. Binding the specific immunomagnetic beads to the target bacteria provides a suitable electric resistance change in the treated filter. The developed tool could detect different live and killed bacteria in any media. According to the bacterial concentrations in the tested samples, the electrical resistance showed more changes at the 200 k, 2 m, or 20 m detection points. In addition, the higher concentration of the target bacteria caused a higher change in the electrical resistance in all of the detection points. These variances could be used for quantification of the immunoelectric results. For more information, see Appendix S1. The milk samples are rich of biological elements, like fats, carbohydrates, proteins, and enzymes. So, the successful detection of the bacteria in bovine's mastitis milk can be a sign of hope for applying the current methods for tracing pathogenic bacteria in other samples.
FUNDING INFORMATION
This study was financially supported by Shahid Chamran University of Ahvaz, Ahvaz, Iran.
CONFLICT OF INTEREST STATEMENT
The authors declare to have no conflict of interest.
Supporting information
Appendix S1
ACKNOWLEDGMENTS
The authors thank Mohammad Javad Zaki for her kind cooperation in designing the immunoelectric parts and Gholamabass Khosravi for consulting in the field of electronics.
Mohammad K, Meysam M, MohammadNasir G, Darioush G. Detection of bacteria causing mastitis in cows with immunoelectric tool. J Clin Lab Anal. 2023;37:e24939. doi: 10.1002/jcla.24939
DATA AVAILABILITY STATEMENT
The datasets used and/or analyzed during the current study are available as supporting information and 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
Appendix S1
Data Availability Statement
The datasets used and/or analyzed during the current study are available as supporting information and from the corresponding author.