A Systematic Review of the Recent Techniques Commonly Used in the Diagnosis of Mycoplasma bovis in Dairy Cattle

Early detection of Mycoplasmal mastitis is greatly hampered by late seroconversion, slow growth of Mycoplasma organisms, intermittent shedding, and the high cost of diagnostic tests. To improve future diagnostic development, examining the available techniques is necessary. Accordingly, the present study systematically reviewed M. bovis diagnostic studies published between January 2000 and April 2023 utilizing the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol. The protocol registration was performed according to the Open Science Framework (osf.io/ug79h), and the electronic search was conducted in the World Catalog, Mendeley, ProQuest, ScienceDirect, Semantic Scholar, PubMed, Google Scholar, Prime Scholar, and PubMed Central databases using a Boolean operator and inclusion and exclusion criteria. Of the 1194 pieces of literature retrieved, 67 studies were included. Four broad categories of up to 16 diagnostic approaches were reported: microbial culture, serological, DNA-based, and mass spectrometry. Overall, DNA-based techniques were the most published (48.0%), with recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) as the most promising user-friendly, equipment-free techniques. On the other hand, mass spectrometry was reported as the least utilized (2.9%) given the high equipment cost. Though costly and laboratory-allied, DNA-based techniques, particularly PCRs, were reported as the most rapid and specific approach.

Mycoplasma bovis are relatively small with a genome size of ~953 kbp [11] and a low GC ratio and lack of a cell wall [12,13].Because of their small genome, Mycoplasma cannot perform critical metabolic pathways, thus leading to a parasitic and demanding lifestyle.. Their lack of a cell wall makes them resistant to β-lactams and other antimicrobial compounds, thereby limiting the effectiveness of antibiotic treatments [14].M. bovis infection spreads quickly, primarily through animal-to-animal contact.Still, it can also spread through contact with personnel or equipment, airborne transmission, and during artificial insemination, rendering them the most contagious mastitis-causing Mycoplasma spp.[15,16].
Mycoplasma mastitis is considered untreatable with substantial negative impacts on milk production [17] and overall cow weight gain [18], which amounts to severe economic losses to producers.Losses due to mastitis caused by M. bovis in the United States were estimated at USD 108 million per year with herd infection rates of up to 70% [19,20].In 2014, an estimated 99.7% of the dairies in the United States had mastitis, and the average herd prevalence of clinical mastitis was 24.3% [21].In California alone, M. bovis was associated with up to 52.2% of Mycoplasma mastitis [22].
Considering that M. bovis mastitis lacks effective treatment, control of the infection in a herd solely relies on early identification, isolation, and culling of infected animals.Unfortunately, the clinical and pathological signs of M. bovis-infected cattle are non-specific [23], thereby complicating its diagnosis.Thus, rapid, sensitive, and accurate animal testing is needed to control potential outbreaks.To date, microbial cultures, polymerase chain reactions (PCRs), and serological diagnosis using an ELISA remain the principal techniques employed in M. bovis detection [24].However, the economic losses caused by M. bov is diagnosis and the need for user-friendly, field-applicable diagnostics requires an extensive review of the available techniques.Previous reviews on M. bovis diagnosis have focused different aspects including transmission and detection [25], control [17], mastitis importance [26], epidemiology [27], occurrence and control [23], diagnosis and control [28][29][30][31], and virulence and the host immune response in infected cattle [30].Such reviews should have systematically processed the most recent M. bovis diagnostic techniques in much detail.Thus, this study systematically examined recent diagnostic techniques for M. bovis, including their benefits, accuracy, and limitations, to aid in the development of future diagnostics.

Literature Search Strategies
The present systematic review was executed in compliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) [32] protocols registered in Open Science Framework (osf.io/ug79h).Here, the Boolean operator [(diagnos*) or (detect*) AND ("bovine mastitis") or ("mycoplasma bovis mastitis") or ("bovine mycoplasmosis")] was used to carefully search World Catalog, Mendeley, ProQuest, ScienceDirect, Semantic Scholar, PubMed, Google Scholar, Prime Scholar, and PubMed Central databases for literature published in English between January 2000 and April 2023.

Eligibility Criteria
To ensure that only relevant studies were included, the articles were first checked for any duplicates using their titles.The abstracts of potential articles were then screened to determine their eligibility.Only original studies that reported on the diagnosis of M. bovis and that met strict inclusion criteria were included.Review articles, both published and unpublished, were not considered.Articles related to other types of mastitis and those that were not available in English were also excluded.Finally, articles that did not provide information on M. bovis detection or whose full text was not available at the time of the search were excluded.

Data Extraction and Appraisal
A multiple-assessment approach across authors was used to identify, screen, and select studies based on PRISMA guidelines [32].During the research process, regular discussions were held among the team while iterating through screening, analysis, and synthesis.To evaluate the literature, the Guide Evaluation of Qualitative Research Studies (GEQRS) was utilized [33].The assessment also utilized Joanna Briggs Institute's critical appraisal checklist for studies reporting on prevalence data fields [34].Data were then extracted using the guidance for conducting systematic scoping reviews [35,36].To check for the consistency and reliability of studies, two authors conducted the quality appraisal approach [32,37].Articles that met less than 70% of the assessment criteria signified high bias or risk and were dropped [37].Table 1 presents a summary of the articles that were included, showing the type of biological sample, diagnostic methods used, the detection unit, percentage of M. bovis detected, as well as the country of origin, authors, and year of the study.

Results and Discussions
A total of 1194 articles were obtained through the database searches.After the removal of duplicates and assessing the full-text articles based on the inclusion and exclusion criteria, only 67 primary studies were qualified (Figure 1).The highest proportion of these studies were conducted in the United States (11.9%), followed by Australia (8.9%), and India (7.4%).Most of the studies included in the analysis utilized milk samples for the diagnosis of M. bovis (41.8%), while a few used sera (17.9%), and the least used sample type was serum (1.5%).

Results and Discussions
A total of 1194 articles were obtained through the database searches.After the removal of duplicates and assessing the full-text articles based on the inclusion and exclusion criteria, only 67 primary studies were qualified (Figure 1).The highest proportion of these studies were conducted in the United States (11.9%), followed by Australia (8.9%), and India (7.4%).Most of the studies included in the analysis utilized milk samples for the diagnosis of M. bovis (41.8%), while a few used sera (17.9%), and the least used sample type was serum (1.5%).To accurately diagnose M. bovis mastitis and differentiate it from other types of mastitis, laboratory testing, such as bacterial culture and identification, or molecular tests, such as a PCR, is required.Unfortunately, the expense of molecular tests makes them impractical for routine use.Additionally, the lengthy process for bacterial identification and turnaround time makes it less effective for guiding management decisions.The diagnosis process is further complicated by intermittent bacterial shedding and subclinical infection states.The present study evaluated over 14 diagnostic methods broadly categorized into To accurately diagnose M. bovis mastitis and differentiate it from other types of mastitis, laboratory testing, such as bacterial culture and identification, or molecular tests, such as a PCR, is required.Unfortunately, the expense of molecular tests makes them impractical for routine use.Additionally, the lengthy process for bacterial identification and turnaround time makes it less effective for guiding management decisions.The diagnosis process is further complicated by intermittent bacterial shedding and subclinical infection states.The present study evaluated over 14 diagnostic methods broadly categorized into bacterial culture, serology, DNA-based techniques, and mass spectrometry.Most of the studies included in the review (24 out of 67) were conducted between 2020-2023, followed by those reported between 2015-2020 (21 of the 67).The least number of studies were encountered in the early 2000s (6 out of 67) (Figure 2).Overall, DNA-based techniques were the most reported technique (48.0%) for the diagnosis of bovine mastitis, followed by serology (26.5%), while mass spectrometry was the least utilized technique (2.9%).

Microbial Culture
In this study, although culturing was once considered the gold standard for M. bovis detection, it was the third most reported technique, likely due to the evolution of new techniques [23].In addition, the culture method is comparatively inexpensive [5] with a contemporary cost range of USD 5 to USD 6.5 depending on the laboratory used [82].According to a previous study, the culture method successfully detected 98% of M. bovisinfected caseo-necortic lung tissues in Canadian feedlot beef calves [93].Similarly, studies by Sickles et al., 2000 andWilson et al., 2009 reported the successful detection of M. bovis in 13.7% and 3.2% bulk tank milk samples, respectively [90,100].Since the culture method can detect the viable Mycoplasma species with a high specificity and sensitivity (101-102 CFU/mL), the technique remains vital for the laboratory diagnosis of M. bovis [29,102].Moreover, the centrifugation of milk samples and plating the resuspended pellets instead of direct sample plating further improves the detection of Mycoplasma spp.by four-fold [103].
A major limitation of the culture method is that it can only identify Mycoplasma organisms to the genus level [104].Moreover, the culture method is a laborious and timeconsuming process, as growth is only typically observed after three days with a characteristic "fried-egg" appearance [105].Negative plates should be reexamined after 7 days of incubation [104,106].Unfortunately, due to the highly contagious nature of M. bovis and the long culture period, it is challenging to implement timely responses in cows with positive culture results.This delay can lead to the spread of the disease to other animals Overall, DNA-based techniques were the most reported technique (48.0%) for the diagnosis of bovine mastitis, followed by serology (26.5%), while mass spectrometry was the least utilized technique (2.9%).

Microbial Culture
In this study, although culturing was once considered the gold standard for M. bovis detection, it was the third most reported technique, likely due to the evolution of new techniques [23].In addition, the culture method is comparatively inexpensive [5] with a contemporary cost range of USD 5 to USD 6.5 depending on the laboratory used [82].According to a previous study, the culture method successfully detected 98% of M. bovis-infected caseonecortic lung tissues in Canadian feedlot beef calves [93].Similarly, studies by Sickles et al., 2000 andWilson et al., 2009 reported the successful detection of M. bovis in 13.7% and 3.2% bulk tank milk samples, respectively [90,100].Since the culture method can detect the viable Mycoplasma species with a high specificity and sensitivity (101-102 CFU/mL), the technique remains vital for the laboratory diagnosis of M. bovis [29,102].Moreover, the centrifugation of milk samples and plating the resuspended pellets instead of direct sample plating further improves the detection of Mycoplasma spp.by four-fold [103].
A major limitation of the culture method is that it can only identify Mycoplasma organisms to the genus level [104].Moreover, the culture method is a laborious and time-consuming process, as growth is only typically observed after three days with a characteristic "fried-egg" appearance [105].Negative plates should be reexamined after 7 days of incubation [104,106].Unfortunately, due to the highly contagious nature of M. bovis and the long culture period, it is challenging to implement timely responses in cows with positive culture results.This delay can lead to the spread of the disease to other animals within the herd.M. bovis has very specific growth requirements and is a slow-growing organism that can easily be overgrown by contaminating bacteria.Growth contamination can, however, be avoided by supplementing the media with antibiotics.The principal media commonly used for the detection of M. bovis infections include Eaton's [106], Hayflick's [107], and modified PPLO [108].These media require good laboratory settings, such as a carbon dioxide incubator, as well as a skilled workforce for the successful isolation of M. bovis.
Ordinarily, the detection of M. bovis infection in a culture requires the cattle to be shedding viable organisms.Even so, studies have shown that there is intermittent shedding in chronic and subclinical mycoplasma mastitis cases, with up to 56 days without shedding in cattle with chronic mastitis [27,29,109].This calls for alternative techniques like serology that do not rely on the shedding of viable organisms by the cattle.Additionally, culture methods are unable to differentiate between closely related species.Mycoplasma species cannot be distinguished from Acholeplasma, a species considered non-pathogenic, on a modified Hayflick medium without additional tests.Essentially, aide tests, such as digitonin or nisin disk diffusion assays, or PCRs are inevitable [110].Overall, microbial culture techniques have limitations that affect their effectiveness and efficiency in diagnosis [111].These limitations include time, field applicability, and accuracy.

Serology
Serological techniques can detect specific antibodies against M. bovis in milk, plasma, or serum samples within two weeks of infection.Since only the antibody response is detected, it is not necessary for the cows to be actively shedding the pathogens at the time of sample collection, unlike with the culture method [18], and the antibody level remains high for several months [112].This particular attribute renders serology a reliable technique in herds where heavy antibiotics use and chronic infections usually impede M. bovis isolation [106].However, serology, just like the culture technique, is laboratory-based and demands trained staff.During this review process, five serological techniques were encountered, and these include, an enzyme-linked immunosorbent assay (ELISA) [43,78], immunohistochemistry (IHC) [113], the direct fluorescent antibody technique (DFAT) [114], an immunobinding test (IBT) [115], sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE,) and immunoblotting [83].

Enzyme-Linked Immunosorbent Assay
This is the most used serological technique for detecting M. bovis-specific antibodies.Most of the published serological studies (70.4%) employed an ELISA in diagnosing M. bovis.The ELISA is mostly utilized in herd-level diagnosis rather than individual animal testing, with results available within two days [27,116].Through the ELISA technique, a large number of samples can be screened; thus, it is ideal for surveillance or biosecurity programs [98].To date, various commercial ELISA kits are available, including Bio X, CHECK-IT M. bovis-Sero, IDEXX, Biovet, and MilA, among others, with the Bio-X M. bovis ELISA kit being the most utilized kit.A study conducted in Turkey [80] showed that the BIO-X M. bovis antibody ELISA kit detected up to 35.4% of the M. bovis-specific antibodies in 127 tracheal swab samples across seven geographically distinct farms in Turkey.Even higher percentages of M. bovis-specific antibodies (38% and 46%) were reported using the same kit in Nigeria [78] and Australia [70].
As much as the ELISA technique is generally less labor-intensive and time-consuming than culture methods, seroconversion may take 2-3 weeks before antibodies can be detected and may lead to false negative results [117].In addition, uncertainty due to cross-reactivity with other closely related organisms decreases ELISA specificity [118] and hence the need for complementary techniques for an accurate diagnosis.Additionally, the presence of anti-M.bovis antibodies does not mean the animal has or is shedding viable pathogens.Still, the presence of antibodies could indicate infection at a site other than the mammary gland or could be a result of maternal or natural antibodies [17,27].It is therefore recommended that the results of the ELISA be used in addition to DNA-based techniques for bulk-tank milk testing.While bulk-tank sampling may achieve fewer false positives, it decreases the sensitivity of the test to about 43.5%, making it difficult to interpret the results and inform appropriate action for individuals.However, it can still be useful for monitoring herd health [119].

SDS-PAGE and Western Blotting
The antigenic structure of mycoplasma strains can be compared using SDS-PAGE and western blotting.This technique can also reveal the host animal's humoral immune response patterns [120].The technique was first utilized to identify 34 isolates of M. bovis with a high degree of similarity [102].Later, potential M. bovis antigenic proteins were identified after separating them on sodium dodecyl-sulfate polyacrylamide gel electrophoresis [84].The same technique was used to distinguish M. bovis from M. agalactiae based on their native protein patterns on the gel [83].Their study unveiled seven major immunogenic cross-reactive proteins and two important non-cross-reacting species-specific polypeptides (25.50 and 24.54 kDa) in M. agalactiae and M. bovis, respectively.This reaffirmed earlier reports that western blotting might address the cross-reactivity drawbacks of techniques like ELISAs [121].

Immunobinding Test
The immunobinding test is one of the oldest techniques used to diagnose M. bovis.The technique was first used by Infante Martinez et al. (1990) to detect Mycoplasma species in milk [115].They found the assay highly specific when monoclonal antibodies were incorporated.Later, the technique was improved to detect M. bovis in 42.3% (55/130) of naturally infected milk within just 110 min at the highest level of sensitivity and specificity [97].Since then, the technique has evolved into nitrocellulose paper with monoclonal antibodies.This technique was used to detect M. bovis cultural isolates and swabs from the genitals of artificially infected heifers utilizing the PCR as the gold standard [122].

Direct Fluorescent Antibody Technique
In this technique, fluorescently labeled monoclonal antibodies are made to bind and illuminate the target M. bovis antigens directly [115,123].A study on 120 mammary samples from slaughterhouses in Esrzurum Province, Turkey, Altun and Ozdemir (2019), detected 23.3% of M. bovis in the cattle mammary tissue using the direct fluorescent antibody technique.Similarly, other Mycoplasma species, such as M. californicum, M. bovigenitalium, M. canadense, M. arginini, and M. alkalescens, in milk samples from California dairies were effectively speciated at a low cost using this technique [82].Thus, the technique remains the most preferred cost-effective and specific approach to detect and speciate Mycoplasma spp.However, generating fluorescent antibodies is a laborious and skill-demanding process.

Immunohistochemistry
In immunohistochemistry, M. bovis antigens can be detected in situ using immunochemical analysis after fixing the tissue in formalin and embedding it in paraffin [93,113].A study on 35 commercial dairy herds in southern Brazil detected up to 91.4% of M. bovis in pulmonary sections of Holstein cows using immunochemistry [58].Another study detected 73.9% of M. bovis antigens in 23 calves across southern Spain using the same technique.However, a lower value (18.2%) was reported from the lung tissue of 87 bovine carcasses from Indian farms showing visible pneumonic lesions at necropsy [64].Although this serological technique is considered in situ and hence worthwhile over the culture technique, it is still labor-intensive and costly to generate fluorescent antibodies.

DNA-Based Techniques
M. bovis detection is laborious, time-consuming, and difficult to perform.Moreover, the detection of M. bovis through serological methods is challenging due to potential crossreactivity.Therefore, DNA-based techniques have become the favored diagnostic approach.DNA-based techniques, particularly the polymerase chain reaction (PCR) allows for the rapid and specific detection of M. bovis [124].The PCR was the most used diagnostic technique (81.6%) in this investigation, likely due to these properties (Figure 3).The PCR technique offers faster diagnosis compared to culture and serological methods, with results available in just a few hours [102], thus allowing for prompt action, such as the removal of infected cows from the herd.Further, PCR methods can specifically amplify M. bovis DNA, enhancing the identification of Mycoplasma species [125].Moreover, more than one species of Mycoplasma as well as non-cultivable or unknown species can be detected when conventional PCR products are run through denaturing gradient gel electrophoresis [126], a property comparable to microarrays [127].The PCR-DGGE method effectively detected 9.3% M. bovis uvrC genes in 713 nonpharyngeal swabs of Polish cattle [24], thus rendering it an effective and accurate technique.
thogens 2023, 12, x FOR PEER REVIEW 9 M. bovis detection is laborious, time-consuming, and difficult to perform.Moreo the detection of M. bovis through serological methods is challenging due to potential cr reactivity.Therefore, DNA-based techniques have become the favored diagnostic proach.DNA-based techniques, particularly the polymerase chain reaction (PCR) all for the rapid and specific detection of M. bovis [124].The PCR was the most used diag tic technique (81.6%) in this investigation, likely due to these properties (Figure 3).PCR technique offers faster diagnosis compared to culture and serological methods, results available in just a few hours [102], thus allowing for prompt action, such as removal of infected cows from the herd.Further, PCR methods can specifically amp M. bovis DNA, enhancing the identification of Mycoplasma species [125].Moreover, m than one species of Mycoplasma as well as non-cultivable or unknown species can be tected when conventional PCR products are run through denaturing gradient gel elec phoresis [126], a property comparable to microarrays [127].The PCR-DGGE method fectively detected 9.3% M. bovis uvrC genes in 713 nonpharyngeal swabs of Polish c [24], thus rendering it an effective and accurate technique.One drawback of the PCR is that non-viable bacteria can still be detected [128].other drawback associated with the PCR is the high cost involved.Nonetheless, sam pooling has been suggested as a cost-saving approach [57,59,82].Over time, the meth used to diagnose M. bovis with a PCR have evolved.These advancements include the ventional singleplex PCR and multiplex, nested, and real-time PCRs, but they often c with additional costs.

Conventional PCR
The conventional PCR involves in vitro amplification of unique specific DNA tar using sequence-specific oligonucleotide primers and heat-stable polymerase enzy [129].Several M. bovis DNA targets have been reported, and they include uvrC, 16S rR gyrB, polC, 16S-23S rRNA ITS, oppD, vspB, and gltX, among others.In the present stu One drawback of the PCR is that non-viable bacteria can still be detected [128].Another drawback associated with the PCR is the high cost involved.Nonetheless, sample pooling has been suggested as a cost-saving approach [57,59,82].Over time, the methods used to diagnose M. bovis with a PCR have evolved.These advancements include the conventional singleplex PCR and multiplex, nested, and real-time PCRs, but they often come with additional costs.
Additionally, a conventional PCR variant (the multiplex PCR) in which more than a pair of primers is utilized for simultaneous amplification of multiple target sequences in a single reaction tube [130,131] was reported.Still, uvrC genes were the most preferred target given that 16S rRNA genes display a low level of variation to differentiate between closely related Mycoplasma species, like M. bovis and M. agalactiae [132].The multiplex PCR was employed in the detection of M. bovis genes as well as in distinguishing it from M. agalactiae isolated from milk and nasal swabs based on the multiplex PCR amplified products [95].In a study from Argentina, Neder and colleagues (2021) used two sets of primers (MBOUVRC2-L and MBOUVRC2-R) and could detect 7.9% (n = 38) of M. bovis genes in the bulk tank milk samples amidst other Mycoplasma species, like M. californicum, M. canadense, and M. leachii.
To further improve both the sensitivity and specificity of the conventional PCR, the nested PCR evolved [133].Unlike the multiplex PCR, a nested PCR utilizes two pairs of amplification primers in two successive rounds of a PCR [26,133,134].In their study on field milk samples from Iowa farms, USA, Pinnow et al. (2001) detected 49.1% M. bovis genes in 53 milk samples using the nested PCR to a sensitivity of 5.1 CFU/mL.Later, a semi-nested PCR was reported to have detected M. bovis genes in milk (27.5%), nasal (30.0%), conjunctiva (12.5%), and vaginal (2.5%) samples during their first test on 40 milking Friesian-Holstein dairy cattle in Australia [96].

Real-Time PCR
The real-time PCR quantifies the amplified PCR products based on different fluorogenic DNA probes [135].It has extra benefits over the conventional PCR as a faster and more sensitive alternative with no need for post-reaction handling.The sensitivity and specificity of the real-time PCR for mastitis detection may reach 100% [136,137].However, these advantages come at a much higher financial expense, as qPCR testing for three pathogens can cost between USD 19-50 compared to a culture cost of USD 5-6.50 [82].Should a dairy wish to run samples in-house (an on-farm PCR) to cut costs, it would still require access to specialized equipment and trained personnel, which are equally costly.In addition, an on-farm PCR is prone to high contamination that can cause false positives or a high threshold for the detection limit that can increase the possibility of false negatives.
Several commercial qPCR kits are currently available for the detection of M. bovis.PathoProof Mastitis Major-3 by Thermo Fisher Scientific is one of the commercially developed qPCR kits capable of detecting M. bovis alongside two other contagious mastitis pathogens (S. aureus and S. agalactiae).The bacto-type Mastitis HP3 PCR kit from QIAGEN is another highly sensitive and specific test for the identification and differentiation of DNA from three major mastitis-causing pathogens (M.bovis, S. agalactiae, and S. aureus) in milk samples (quarter milk samples, pool, or bulk milk).The Pneumo4B and Pneumo4V qPCR was developed in 2020 for the detection of M. bovis in the tracheal aspirate samples from calves [138].Other commercial kits include the VetMAX™ MastiType Myco8 Kit (ThermoFischer, Warrington, United Kingdom) and Mastit4 (DNA Diagnostics, Risskov, Denmark).
Using a real-time PCR, several studies reported the successful and accurate diagnosis of M. bovis mastitis worldwide.In India, Behera et al. (2018) utilized a SYBR green dyebased real-time PCR assay targeting the uvrC gene for the diagnosis of M. bovis in milk and lung tissue.Recently, targeting polC genes, Becker et al. (2020) reported 51.0% of M. bovis polC genes as positive samples in 251 nasal swabs of calves in western France.The same gene was utilized in a real-time PCR that detected 58% of M. bovis in 351 bronchoalveolar lavage fluids (BALF) from calf farms in Algeria [42].Thus, the extensive use of the real-time PCR could unlock new opportunities for the control of diseases caused by M. bovis provided the costs involved are cut.Developing new qPCR assays remains one of the attempts towards cost reduction.Chauhan and colleagues (2021) developed a multiplex qPCR to simultaneously detect M. bovis, A. laidlawii, and several Mycoplasma species, like M. californicum, M. bovigenitalium, M. canadense, M. arginini, and M. alkalescens.This assay, developed to target the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S-23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii, could detect and distinguish M. bovis from other prevalent Mycoplasma spp.and the non-pathogenic A. laidlawii in the milk samples collected from California dairy farms.However, sample preparation involving a QIAGEN DNeasy Blood and Tissue Lysis kit could potentially soar the cost of running such assays [139].

Recombinase Polymerase Amplification
Interferences from proteins, fats, and ions greatly hinder the quantitative detection of bacteria using real-time PCRs [140,141].One of the remedies is protease pre-treatment prior to direct detection.Rossetti and colleagues [142] developed a real-time PCR assay targeting the uvrC gene to directly detect M. bovis from milk and tissue samples with highly reduced interference.Such an approach sheds light on the efforts towards the development of robust and effective testing, which is currently lacking.Simple yet robust, the recombinase polymerase amplification (RPA) technique is yet another promising isothermal DNA amplifying assay with reduced external interference for possible rapid field-applicable tests.
By targeting uvrC genes, RPA directly detected 36.9%. of M. bovis genes after 15 min incubation at 39 • C and 5 min visualization without any interference.The 65 milk samples for this validation were from the eight different dairy farms in Baoding and Hengshui, Hebei Province, China [52].In the same way, RPA was combined with a lateral flow dipstick (LFD), and the assay successfully detected M. bovis DNA in 30 min at 39 • C with a detection limit of 20 copies per reaction when compared to a real-time quantitative PCR (qPCR) assay [71].Such findings open new frontiers for the exploration of a simple and cost-effective alternative to the real-time PCR.

Loop-Mediated Isothermal Amplification
Just like RPA, another potential pen-side test for the detection of M. bovis is the loopmediated isothermal amplification (LAMP) assay [60,63,66,76,88,143], though extensive validation for its reliability awaits.As a simple and cost-effective assay, LAMP is a rapid test with a reaction taking less than 2 h.Moreover, there is no need to have expensive laboratory equipment, as a single temperature is required [144].Accordingly, LAMP is perceived as a potential cheap diagnostic tool.However, high background signals of some assays, vulnerability to cross-contamination/DNA carryover, and the complex primer design may compromise the specificity, sensitivity, and simplicity of the assay, respectively [145,146].
To reduce background signals and cross-contamination, various DNA purification kits have been employed.They include the MoBio DNA extraction kit [63], QIAGEN DNeasy Blood and Tissue kit [66], TIANamp Genomic DNA kit [143], and Procedure for UltraRapid Extraction (PURE) kit [60].However, such interventions attract additional costs to the technique, making it lose its cost-saving attribute.

Pulse Field Gel Electrophoresis
During pulse field gel electrophoresis (PFGE), M. bovis genomic DNA is first extracted and then digested using a restriction enzyme.Later, the digested products can be assessed on an agarose gel by subjecting it to an electric field that periodically changes direction to aid the separation of the larger DNA fragments [19].Through this technique, M. bovis was fingerprinted in 34.0%(n = 151) of infected milk samples and mucosal swabs of lactating cows [89].Similarly, PFGE detected M.bovis in French calf feedlots [86] and Danish cattle [101], among others.While PFGE can identify bacteria up to the strain level, the technique is time-consuming and requires specialized skills.

Mass Spectrometry
Following the cultural isolation, a more rapid technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be applied to detect M. bovis [147].The principal strength of this technique is based on its ability to only detect viable bacteria, implying the animal has active rather than historic infections [148].It is worth noting that, for rapid and effective detection of M. bovis using MALDI-TOF MS in routine veterinary laboratories, culture enrichment is encouraged.Enrichment enhanced the identification of 38.0% of M. bovis from 100 bronchoalveolar lavage fluid (BALF) after 72 h of enrichment [54].Likewise, McDaniel and Derscheid (2021), accurately identified all eight isolates of M. bovis using M. arginini and M. alkalescens as controls to evaluate the specificity of MALDI-TOF MS.The isolates were first cultured in pleuropneumonia-like organism (PPLO) broth with horse serum (University of California-Davis, Davis, CA) 4 days prior to use [51].In the most recent attempt, Thompson et al. (2023) combined MALDI-TOF with machine learning as an alternative diagnostic tool to detect the high somatic cell count (SCC) and subclinical mastitis in dairy herds around Texas, USA.Their study involving 100 milk samples showed a high sensitivity and specificity [38].As much as the technique is reliable, rapid, and cost-effective for routine identification of unknown Mycoplasma isolates, it suffers from the limitations of the culture technique.Additionally, MALDI-TOF MS is a recent technique, and only a limited library for Mycoplasma species exists.

Conclusions and Future Perspectives
The present study systematically reviewed several M. bovis mastitis diagnostic approaches.The bacterial culture method requires a specialized laboratory, technical staff, and time-consuming procedures.However, bacterial cultures provide discrete colony isolates for DNA-based and mass spectrometry diagnostics.On the other hand, serological techniques are faster than cultures but have low specificity due to cross-reactivity.The 2-3 weeks seroconversion period also increases the chances of false negative results.Hence, it is advisable to complement serology with other DNA-based techniques to achieve the accurate detection of early and chronic M. bovis.DNA-based assays, especially PCRs, are the most used technique due to their quick and accurate results, allowing for timely intervention.As much as the PCR has the potential to overcome the limitations of cultures and serology, it is costly and requires specialized equipment that limits its use in point-of-care field settings.While combining two techniques for diagnosing M. bovis is recommended, isothermal amplification technology (IAT) has shown higher sensitivity and specificity as a stand-alone technique with results obtained within two hours.Collectively, isothermal techniques just like any new diagnostic test should be validated before clinical use.

Figure 1 .
Figure 1.PRISMA flow diagram on recent techniques for M. bovis detection.The 1194 identified documents were screened and appraised, from which 67 of them were included in this study.All studies were accessed through an electronic database search.

Figure 1 .
Figure 1.PRISMA flow diagram on recent techniques for M. bovis detection.The 1194 identified documents were screened and appraised, from which 67 of them were included in this study.All studies were accessed through an electronic database search.

Figure 2 .
Figure 2. The number of studies by years.The number of studies on detection of M. bovis increased since 2000 with the highest number (24) recorded between January 2020 and April 2023.The least number of studies (06) were between 2000-2004.

Figure 2 .
Figure 2. The number of studies by years.The number of studies on detection of M. bovis increased since 2000 with the highest number (24) recorded between January 2020 and April 2023.The least number of studies (06) were between 2000-2004.

Figure 3 .
Figure 3. Distribution of DNA-based techniques encountered in the diagnosis of bovine myco mosis.RPA, Recombinase Polymerase Amplification; PCR, Polymerase Chain Reaction; LA Loop-mediated Isothermal Amplification; PFGE, Pulse Field Gel Electrophoresis.PCR techn was the the most utilized DNA-based technique (82.0%), followed by LAMP (10.0%).

Table 1 .
Recent techniques reported in the detection of M. bovis.Sixty-seven diagnostic studies utilizing various biological samples were explored.The included studies were those published between January 2000 and April 2023.