Basic concepts, recent advances, and future perspectives in the diagnosis of bovine mastitis

Abstract

Mastitis is one of the most widespread infectious diseases that adversely affects the profitability of the dairy industry worldwide. Accurate diagnosis and identification of pathogens early to cull infected animals and minimize the spread of infection in herds is critical for improving treatment effects and dairy farm welfare. The major pathogens causing mastitis and pathogenesis are assessed first. The most recent and advanced strategies for detecting mastitis, including genomics and proteomics approaches, are then evaluated . Finally, the advantages and disadvantages of each technique, potential research directions, and future perspectives are reported. This review provides a theoretical basis to help veterinarians select the most sensitive, specific, and cost-effective approach for detecting bovine mastitis early.

INTRODUCTION

Approximately six billion people, over 80% of the world’s population, are regular consumers of liquid milk and other dairy products. The global dairy industry was estimated to be US $330 billion in 2014 [1]. This dramatic increase in global population will increase the demand for the dairy sector. The global food demand is predicted to double by 2050. The United Nations (UN) expects the world’s population to increase from 7.6 billion to 8.6 billion in 2030, 10 billion in 2050, and 11.2 billion in 2100 [2]. In 2007, Wen Jiabao, the then-Premier of the People’s Republic of China, said, “I have a dream to provide every Chinese, especially children, sufficient milk each day.” There are an estimated 1.4 billion people in China. Thus, much more milk will be needed to achieve Wen Jiabao’s dream.

Nevertheless, mastitis seriously affects milk production in China, with an estimated incidence of clinical mastitis (CM) at the cow and quarter levels of 8.7 and 3.7%, respectively, and that of subclinical mastitis (SCM) at the cow and quarter levels of 48.8 and 19%, respectively [3]. The inflammatory response of udder tissue is defined as mastitis [4]. According to the severity of inflammation, mastitis can be divided into two types: SCM, which does not cause any apparent changes in milk or udder morphology, and CM, which is accompanied by visible changes in the milk or mammary gland [5] (Fig. 1). CM is considered the tip of the iceberg. Indeed, in most herds, SCM is 15 to 40 times more widespread than CM [6]. Globally, the reported prevalence of mastitis differs according to studies in developed and developing countries, as listed in Table 1. The reported variations are related to management and detection methods. In wealthy nations with the beginning of modern dairy farming, effective methods have been developed to minimize and detect the occurrence of mastitis in cattle herds. These methods are not accessible in low-income countries [3]. Many predisposing factors influence the incidence and prevalence of mastitis: animal age, lactation stages, milking hygiene, environmental conditions, nutrition, and genetics.

Fig. 1. Cattle clinical mastitis. A case with an inflamed right rear quarter in a dairy farm in Hubei province, China.

Table 1. Prevalence of cow mastitis in different regions of the world.

Area		Prevalence of mastitis		References
Worldwide		• The collective prevalence of Staphylococcus, Streptococcus, and E. coli species were 28%, 12%, and 11% worldwide from 156, 129, and 92 studies, respectively.		[150]
		• The pooled prevalence of CM and SCM were 15% and 42%, respectively.		[151]
	Canada	• 40% in British Columbia, 60% in Alberta, approximately 70% in Ontario and Quebec, and up to 90% in Saskatchewan and Nova Scotia.		[152]
	Brazil	• 46.4% for SCM during 2011–2015.		[153]
	Urban and peri-urban areas of Thika, Kenya	• 64% SCM at the cow level and 55.8% at the quarter level.		[154]
	New South Wales, Australia	• 28.9% SCM between January 2006 and June 2009.		[155]
			- 16.1% in the low prevalence category, whereas on farms in the high category was 36.5%
	Central region of Fars province, south of Iran	• Clinical and subclinical mastitis at cow level was 2.2% and 42.5%, respectively.		[156]
	Finland	• SCM prevalence has decreased over recent decades in Finland from 22.3% (1991) and 20.1% (2001) to 19.0% (2010).		[157]
			- Prevalence of chronic subclinical mastitis was 20.4%
	North-West Ethiopia	• 33% for SCM of the quarters and 62% at the cow level.		[158]
	Holeta district, Ethiopia.	• 41.02% SCM between August 2011 and May 2012.		[159]
	Peri-urban areas of Kigali in Rwanda	• 43.1% SCM at the quarter level and 76.2% at the cow level.		[160]
	Urban and peri-urban areas of Kampala, Uganda	• 48.6%–86.2% at the cow level and 19.6%–55.4% at the quarter level for SCM.		[161]
	Rubavu and Nyabihu Districts, Rwanda	• 50.4% for SCM at the cow level.		[162]
	Northwest of Pakistan	• 20% for CM in cattle compared to 11% in buffaloes.		[163]
			- 66% for SCM in buffaloes compared to 53% in cattle
	Jhenaidah, Bangladesh	• For SCM, the figures vary as follows:		[164]
			- 71.9% at the farm level
			- 67.9% at the individual animal level
			- 29.5% at the quarter level
	Rewa district, Madhya, Pradesh, India	• The overall prevalence of SCM was 31.40% on an animal level, 7.85% on a quarter level, and 2.48% on a blind teat level. According to the lactation stage, SCM prevalence,		[165]
			- 36.54% in early lactation
			- 34.38% in late lactation
			- 27.78% in mid-lactation
	India	• The pooled prevalence of CM and SCM were 18% and 45%, respectively.		[151]
	West Littoral Region in Uruguay	• 52.4% for SCM at the cow level and 26.7% at the quarter level.		[166]

SCM, subclinical mastitis; CM, clinical mastitis.

Mastitis is one of the most ubiquitous problems in high-yielding dairy cattle, causing substantial financial losses with a harmful impact on the milk value [7]. The consequential financial losses include functional losses to the udder or mammary gland and downgraded milk value, such as continuous reductions in milk quantity, decreases in milk protein score, and complete cessation of milking. In addition, indirect losses are incurred, such as culling the cattle early, increasing labor costs, selling animals at low prices, extra veterinarian expenses, drug costs, and laboratory and diagnostic costs [8].

This review manuscript critically updates various aspects of bovine mastitis, focusing on its etiological agents, pathogenesis, advances in diagnosis (e.g., nanotechnology, genomics, and proteomics approaches), and the future directions for the early identification of mastitis. The advantages, disadvantages, sensitivity (Se), specificity (Sp), and predictive values of each technique are analyzed.

MAJOR PATHOGENS AND PATHOGENESIS MECHANISM UNDERLYING BOVINE MASTITIS

Bacteria are the most common cause of mastitis in cattle, and more than 150 diverse bacterial species and subspecies have been implicated in the initiation of bovine mastitis [9]. Among them, 95% of the mastitis cases detected worldwide are caused by only 10 groups. Mastitis can be divided into three groups according to the main reservoir, transmission mode, and bacteriological differentiation: environmental or contagious pathogens, major or minor pathogens, and gram-positive or negative organisms [10]. The udders of diseased cows are the main reservoir of contagious bacteria transmitted from cattle to cattle primarily during milking, and they are susceptible to chronic subclinical infections with outbreaks of clinical incidents [11]. The contagious pathogens include Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma. The main environmental pathogens are represented mostly by coliforms (Escherichia coli, Klebsiella spp., and Enterobacter), environmental streptococci (Str. uberis, Str. dysgalactiae, and other Streptococcus spp.), coagulase-negative Staphylococcus (CNS), and other gram-negative bacteria, such as Serratia, Pseudomonas spp., Proteus, and Pasteurella spp. [12].

S. aureus is considered the major pathogen isolated in the case of mastitis. Bortolami et al. [13] reported that the most common organisms isolated from 98 infected Holstein Friesian dairy cattle on four farms were S. aureus, Str. agalactiae, and Str. Uberis, with rates of 41.8%, 12.4%, and 11.9%, respectively. S. aureus has a complex pathogenesis that makes it challenging to treat with conventional therapy, including facultative intracellular parasitism, increasingly severe antimicrobial resistance, biofilm formation, and evasion of the host immune system [14]. Streptococcus species are other bacteria that can be isolated in approximately 20.4% [15] or 27% [16] of mastitis cases. Str. agalactiae is a mastitis-causing pathogen that can only grow and multiply in the udder. On the other hand, it can survive for short periods on hands, teat skin, and parts of milking machines, leading to its spread among cows during milking [17]. In dairy herds, Mycoplasma bovis is considered one of the most common etiological agents of mastitis, with an incubation period of approximately 10–14 days [18].

Environmental mastitis (EM) has been emphasized for a long time. EM is caused by improper sanitation during or between milking processes, such as improper cleaning of the milking parlor, inappropriate milking machines, insufficient preservation, and contamination of the milk. More importantly, the environmental pathogens most frequently encountered are environmental streptococci (species of streptococci other than Str. agalactiae) and coliform bacteria. According to the management of dairy farms, other EM-causing pathogens, such as Str. uberis, can be found in bedding, traffic lanes, water troughs, and the outdoor environment, including soil and grass [19]. Str. uberis is most commonly associated with moderate mastitis without systemic signs [20]. In addition, coliform bacteria, such as E. coli and Klebsiella spp., are ubiquitous in the bovine environment and are among the most common mastitis-causing pathogens [21]. In dairy cows, coliform mastitis can range from a mild disease with short duration to a severe, per-acute, life-threatening condition. The severity of coliform mastitis is associated with the degree of production loss and clinical outcome [4].

Non-aureus staphylococci, also called CNS, are minor pathogens causing mastitis commonly found on the teats of cows and the hands of milking personnel [22]. CNS groups comprise over 50 bacteria most frequently isolated from infected glands. This group includes S. chromogenes, S. hyicus, S. simulans, and S. epidermidis [23]. Surprisingly, CNS infections only represent 10% of all clinical cases of mastitis [24]. As a result, CNS infections are usually mild and subclinical, causing a two- to three-fold increase in somatic cell count (SCC). CNS mastitis is more prevalent in primiparous cows than in older cows [25].

Developing successful control programs requires an identification of the mechanisms of infection, proper definitions of the clinical and subclinical states of the disease, proper screening tests, and identification of pathogen-specific characteristics. Therefore, a broad understanding of the pathogenesis of mastitis is critical to discovering appropriate detection methods and treatment measures. Various bacterial strains are the most significant causes of mastitis [26]. Usually, the teat orifice is considered the first line of defense against infections in the mammary gland [27,28]. Once the microorganisms enter the teat, they evade the humoral and cellular immunity of the gland (Fig. 2). These bacteria multiply, proliferate, release toxins, and enhance epithelial cells and leukocytes to liberate chemoattractants [10], oxygen radicals, and acute phase proteins (APPs). These factors stimulate the migration of the circulating effector cells responsible for immunity, especially polymorphonuclear neutrophils (PMNs), to the location of inflammation. The causative agent, immune status of the cow, genetics, stage of lactation, age, and nutritional status influence the magnitude of the inflammatory response [29].

Fig. 2. Schematic diagram of mastitis growth in the inflamed mammary gland. Environmental [182] and contagious microorganisms invade the udder through the teat orifice. They then multiply in the udder, where they are attacked by neutrophils while damaging the epithelial cells lining the alveoli with the subsequent release of enzymes, such as NAGase and LDH. Epithelial cells also secrete antimicrobial compounds. Considerable tissue damage is observed once the immune effector cells begin to combat the invading pathogens [183].

NAGase, N-acetyl-β-D-glucosaminidase; LDH, lactate dehydrogenase enzyme.

Masses of PMNs pass between milk-producing cells into the lumen of the alveolus, increasing the SCC and damaging secretory cells. The PMNs contain bactericidal peptides, proteins, and enzymes in their intracellular granules [30]. The oxidants and proteases produced kill bacteria and some epithelial cells, resulting in decreased milk production and the release of enzymes, such as N-acetyl-b-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) (Fig. 2). PMNs are destroyed by apoptosis after their task is fulfilled. Subsequently, macrophage cells engulf and swallow the remaining PMNs [31]. Dead and destroyed mammary epithelial cells and dead granulocytes are secreted into the milk, elevating the milk SCCs. If the infection persists, internal swelling of the mammary epithelium that is not typically noticeable on a physical examination can occur. In addition, the anatomical structure of the alveoli of the gland can be damaged and misplaced (Fig. 2). Blood might be observed in the milk after extensive damage to the blood-milk barrier, leading to noticeable changes in the shape of the gland, such as external swelling and congestion. Furthermore, other changes in the milk can be observed, including increased conductivity, elevated pH, increased water content, and the presence of detectable flakes and clots [29].

MASTITIS DIAGNOSIS

The diagnosis of CM is frequently based on a mammary gland inspection, the presence of any changes in the physical characteristics of milk, an increase in the number of somatic cells, and the identification of bacteria by indirect tests [32]. In contrast, the diagnosis of SCM is more complicated and relies on direct and many indirect methods for estimating the increase in SCC (Fig. 3) [10]. A model diagnostic analysis must be characterized by satisfactory specificity, sensitivity, speed, safety, simplicity, and cost-reflectiveness. This section outlines the progress and properties of different diagnostic methods.

Fig. 3. Direct and indirect methods of mastitis diagnosis.

SCC, somatic cell count; Hp, haptoglobin; Hb, hemoglobin.

Basic diagnosis

Bacteriological examination

A bacteriological culture (BC) is the gold standard for detecting microorganisms that cause mastitis. It identifies the pathogens based on the colony shape, culture media, staining techniques, and assays, such as hemolysis, potassium oxide (KOH), catalase, asculin hydrolysis, citrate utilization, and Christie, Atkins, Munch, and Peterson (CAMP).

Other automated approaches are used for bacterial identification. For example, a biology company applies panels of biological reactions using carbon sources [33]. The BD phoenix and VITEK 2 systems are based on automated biochemical reactions to enhance the efficacy of the microbial identification products [34,35]. Recently, this method was developed using a fluorescence system to detect the metabolic changes produced by various microorganisms [36]. An accuracy, positive predictive value (PPV), and negative predictive value (NPV)of 98%, 86%, and 99%, respectively, were achieved for the diagnosis of contagious and EM [32]. Se and Sp of the culture at the cow level are considered highly significant as they allow critical decisions about using these techniques for controlling a mastitis diagnosis. For example, the mastitis culture technique had a 99.8% Sp (specificity) for S. aureus with a very low false positive percentage (0.2%) and an 88.4% Se (sensitivity), making it suitable as a gold standard test [37]. BCs, however, have limitations differentiating healthy and ill cattle. For example, the results will be poor if the wrong media are used for the microorganisms; the milk appears normal with no alterations during the intermittent shedding stage, but intramammary infection can occur. Moreover, bacterial cultures are time-consuming and do not detect the level of infection-related inflammation [38].

SCC

Veterinarians and farmers have been using (cow-side or on-site) tests to detect mastitis as they require relatively little training. Somatic cell counting detects milk quality and screens for intramammary infections [36]. The Se, Sp, and accuracy of SCC were 86.60%, 97.76%, and 91.94%, respectively, with a PPV and NPV of 98.33% and 84.52%, respectively [39]. These cells are present in normal milk in small amounts, but their levels might be higher in the milk from diseased cattle, possibly reaching more than 90% [40]. Different opinions about the SCC threshold exist. Schukken et al. [41] reported that the uninfected quarters had approximately 70,000 cells/ml, while the milk quantity decreased when the cells exceeded 100,000 cells/ml. On the other hand, other researchers reported that the level of SCC in mastitic milk was more than 250,000 to 300,000 [42]. Petzer et al. [43] indicated that the optimal thresholds of SCC for composite and quarter milk samples were 150,000 and 200,000 cells/mL, respectively. Although SCC has been used for decades in the diagnosis of mastitis, it has some limitations. For example, it can be affected by age, season, stress, management, parturition, and lactation period [41]. Therefore, these limitations should be considered when interpreting the result.

1) California mastitis test (CMT)

The CMT test is rapid, inexpensive, and simple and is one of the most common techniques used to count somatic cells indirectly in milk samples [41]. This test is the major cow site test used to detect the infected quarter for the following bacteriological examination [26]. Sharma et al. [39] reported that the CMT had Se, Sp, accuracy, PPV, and NPV values of 86.07%, 59.70%, 75.52%, 76.21%, and 74.07%, respectively. Nevertheless, this test has some limitations. It cannot be used for large-scale monitoring. It is only used on the farm for a first approach at the cow level. It is affected by stress, season, and parturition, and its interpretation differs between the samples [43]. The Wisconsin mastitis test (WMT) is a different indirect technique that counts somatic cells in the bulk milk tank using the same reagent as the CMT. The test allows the height of the gel in a tube to be measured and reported in millimeters [44]. The relationship between the electronic SCC and CMT results is not precise because of the variability in SCC values within each CMT score [45]. Therefore, indirect on-farm tests that produce a numeric value for the SCC have been developed to overcome this [46]. Somaticell is a modified WMT that is performed in a few minutes and results in a quantitative outcome. Somaticell uses the same methodology as the WMT but was developed to yield results directly as an equivalent SCC. Rossi et al. [47] carried out a more recent study to estimate the accuracy of Somaticell and assess the agreement between Somaticell and the CMT for diagnosing S. agalactiae in mastitic cattle. They reported that the accuracy of Somaticell (n = 319 quarters) in identifying S. agalactiae- infected quarters was 75.4%, 86.4%, 88.9%, 89.4%, and 91.0% at thresholds of 98,000, 147,000, 205,000, 244,000, and 282,000 cells/mL, respectively. The accuracy of the CMT was 87.6%, 90.7%, 90.8%, and 87.4% at the thresholds of trace, 1, 2, and 3, respectively. At the tested thresholds, the Se and Sp of Somaticell ranged from 94.9% to 99.5% and 48.1 to 87.1%, respectively. The Se of Somaticell at the lowest threshold (69,000 cells/mL) was 99.9%, which is higher than that of the CMT at any tested threshold [47].

2) Electrical conductivity (EC) test

The EC test is an indirect screening test used for SCM detection in many countries. The test measures the EC of milk samples by detecting the concentration of sodium and chloride ions in the samples. The diagnostic value of this test for mastitis is controversial. Some researchers mentioned that EC and bacteriological tests are related. By contrast, other authors reported this method to be inefficiently sensitive [26]. Therefore, this method cannot be used alone to detect mastitis because of its low SP, and it is influenced by the following: age, breed, and lactation stage of the cow; temperature; and pH of the milk [48].

Histopathological evaluation

Histopathology is an important and commonly used tool for evaluating damage to the mammary tissue incurred by bacteriological infections of the gland [29]. The mammary glandular tissue samples can be taken using needles and biopsy pistols, as in an automatic needle jab system [49], or using a vacuum system followed by saline lavage of the biopsied site [50]. The representative histopathological results in bovine mastitis affected by S. aureus are as follows: extensive infiltration of PMNs into the gland; reduced luminal space of the alveoli; necrosis and atrophy of the mammary tissue; and replacement of injured secreting mammary tissue with non-secretory tissue. In a histological evaluation, staining and examination hold a very high clinical significance in medical diagnosis and treatment in almost every field of medicine. A histological examination is the gold standard for diagnosing many pathological diseases, including mastitis. The histochemical analysis of a tissue specimen allows a diagnosis and a determination of the disease severity and prognosis. On the other hand, histological evaluations require a complex process, including tissue sampling, fixation, processing, embedding, sectioning, and staining. Finally, it undergoes analysis through microscopy, and the findings are interpreted by a pathologist [51].

Nucleic acid-based diagnosis

Nucleic acid testing

Molecular assays became the ideal standard for mastitis diagnosis in the last decade. They allow rapid, quantitative, qualitative, and wide-scale diagnosis. These methods are based on nucleic acid detection, such as polymerase chain reaction (PCR). Different types of PCR are used to identify the genome structures of the pathogens causing mastitis: amplification of their DNA fragment for their detection only (conventional PCR), detection and quantification of one pathogen (reverse transcription polymerase chain reaction [RT-PCR]), and detection and quantification of the various pathogens in the same sample (multiplex PCR) [52]. The first two techniques for mastitis detection are conventional PCR [53] and multiplex PCR [54]. Both methods have primers designed for 23S and 16S ribosomal ribonucleic acid (rRNA). Gillespie and Oliver [55] used multiplex PCR to detect S. aureus, Str. uberis, and Str. agalactiae instantaneously using primers rather than 16S and 23S rRNA. Multiplex PCR was first designed to detect four pathogens in a single test and was expanded to detect nine [56] or 11 pathogens [57] in a single reaction. Shome et al. [9] used multiplex PCR to identify ten types of bacteria that cause bovine mastitis simultaneously. In addition, Graber et al. [58] used RT-PCR to detect S. aureus mastitis by targeting the nuc gene. These tests have several merits, such as high Se and Sp, speed, and avoidance of the drawbacks related to culture-based tests. Moreover, they can detect the bacteria in milk samples even in the presence of drug residues and preservatives [57,58]. PCR products can be stored in a refrigerator or freezer for an extended period. In contrast, the lengthy storage of culture Petri dishes leads to dryness of the dish or increases fungal growth [59]. Compared to conventional PCR, RT-PCR is safe because ethidium bromide staining is unnecessary; the PCR yields do not require handling and are cost-effective if many samples are used. RT-PCR can differentiate between clonal outbreaks and multiple strains [59], and its Se and Sp reach 100% [57]. On the other hand, PCR has some limitations. For example, it is usually more expensive than immunoassays, and it has been used with limited access in developing countries [60]. False-positive results can be found when milk residues are in the milking machine, contamination, and colonization of the teat canal. They can also arise when using primers that are not sufficiently specific or when the PCR cycle conditions are changed. In addition, clotted milk samples or the presence of PCR inhibitors in the milk samples can result in incorrect DNA extraction and purification, which can lead to a false-negative result. On the other hand, this issue can be overcome by column purification [61].

The fluorescent in situ hybridization technique is another reliable, less time-consuming technique used to detect pathogens causing mastitis, but it is not used routinely because pretreatment steps are needed to obtain the results, and it has a high detection limit [62]. This technique can be used to detect pathogens in cultures or clinical samples. The method uses a fluorescently labeled oligonucleotide probe that can be bound to a specific region of the target sample of DNA or RNA [62]. Other methods depend on the molecular analysis used to identify the pathogens causing mastitis, including amplified fragment length polymorphism (AFLP), which is used to differentiate various S. aureus strains isolated from cattle [2] and ribotyping [63]. Both are used to identify pathogen species. Restriction fragment length polymorphism (RFLP) [64], multiple-locus variable-number tandem repeat analysis (MLVA) [65], and pulsed-field gel electrophoresis (PFGE) typing [66] have been used to recognize the strains. PFGE is still considered the gold standard, mainly for short-term surveillance [65], while multilocus sequence typing is useful for long-term and global epidemiological investigations [67]. The PCR-RFLP approach has been used for epidemiological investigations of S. aureus strains because it is straightforward, rapid, and highly reproducible [64]. Transfer DNA intergenic spacer length polymorphism analysis and DNA sequencing of housekeeping genes were analyzed to differentiate between the species and strain levels [68]. All of these methods need to recover isolates by microbiological culture except for AFLP [2], RFLP [64], and PFGE [66], which can be performed directly from milk samples.

Genomics and proteomics approaches

In recent years, with the ongoing innovation of sequencing technologies, omics technology has been extensively utilized in animal husbandry [69]. Currently, the high-throughput profiling of differentially significant expressed proteins produces abundant information on many diseases. This method is a powerful tool for examining the underlying mechanism, which is a consequence of the rapid development of technologies such as genomics, transcriptomics, and proteomics [70].

1) Genomics approaches

Investigation of specific circulating endogenous non-coding RNA molecules called microRNA (miRNA) is well known because they play a role in genomics and biological processes, including the pathogenesis of inflammatory diseases. This method can recognize pathogens in the early stages of udder infections because miRNAs play essential roles in regulating protein-encoded gene expression that is increased during inflammation [71]. Different kinds of miRNAs are present in milk and are characterized by several merits that make them potential markers for mastitis diagnosis. This type of RNA is unaffected by acidity, room temperature, freezing, or thawing, and it is not digested by RNAse [72]. The expression of miRNAs differs in milk exosomes [73], monocytes [74], epithelial cells, and udder tissue [75]. This expression can be measured by Q PCR and Quan studio 3D digital PCR, which has a higher Se and more reproducibility than Q PCR. Lai et al. [76] showed a higher correlation in the miRNA expression in milk measured by both methods. Bovine mastitis resulted in the differential expression of 25 miRNAs in the mastitic milk samples [77]. Some researchers estimated that the expression profile of at least 14 bovine miRNAs fluctuates according to the health status of the tissue. Between them, the miRNAs 146a and 223 levels increase in early inflammation during mastitis. Therefore, these RNAs can help researchers predict early inflammation of the glands [56]. Tzelos et al. [78] reported that miR-223 could discriminate quarters with early inflammatory changes with high accuracy. Lai et al. [76] showed that several miRNAs had good PV and Se, and Sp values of more than 80% in the differentiation between normal and mastitic milk. The Se and Sp of miR-146a, miR-222, miR-155, miR-383, and miR-21 were as follows:88% and 100%, 94% and 93%, 94% and 90%, 88% and 83%, and 82% and 89%, respectively. On the other hand, Lai et al. [76] suggested downregulation in the miR-26b and miR-29b levels in the milk of mastitic cows compared with healthy cows.

Microarray technology has become an important method for evaluating thousands of genes expressed in tissue. Understanding the biological roles of encoded proteins and the systems of protein interactions in the expression of gene patterns is important. Microarray technology depends on hybridizing different types of target genes overloaded on microarray chips. It imagines them after exposure to the complementary DNA (cDNA) probe bound with chemiluminescent or fluorescence stains [79]. Vidic et al. [80] reported that more than seven pathogens-causing mastitis could be detected in a single reaction by developing multiplex biochips. Lee et al. [81] showed that six field strains for four pathogens can be identified and evaluated in a single milk sample in less than three hours through PCR and a nucleic acid microarray immunoassay.

Hoque et al. [82] predicted the potential dynamic changes in the microbiome composition and the genetic characteristics of the diverse pathogenic forms of mastitis, including CM, recurrent mastitis (RCM), and SCM. Twenty milk samples (five CM, six RCM, four SCM, and five H) were subjected to whole metagenome sequencing (WMS) to elucidate the dynamics, relationships, and pertinent metabolic processes of the microbiome. Four hundred and forty-two bacterial genomes with distinctly different microbiome compositions were obtained from WMS data mapping. In addition, Hoque et al. [82] discovered a variety of genetic characteristics specific to microbes, including 333, 304, 183, and 50 virulence factor-associated genes and 48, 31, 11, and six antibiotic resistance genes in CM, RCM, SCM, and H-microbiomes, respectively. In particular, various metabolic pathways and functional genes linked to the pathogenesis of mastitis were identified [82].

2) Proteomics approaches

Proteomic studies have been pivotal in investigating the genes and proteins linked to the virulence of mastitis-causing pathogens [66]. There are many advances in proper proteomics techniques, such as mass spectrometry (MS) and two-dimensional gel electrophoresis (2D-GE), leading to the recognition of several novel types of proteins related to mastitis [83]. Smolenski et al. [84] used “direct liquid-chromatography tandem MS and 2D-GE” followed by matrix-assisted laser desorption ionization time-of-flight (MALDI) time of flight (TOF) MS analysis of distinct protein spots in mastitis and control milk samples. Only in the infected samples did six chaperonins play an essential role in pathogen identification and act as novel markers for mastitis [65]. 2D-GE is the core technology that detects differences between the protein expression level and modification after translation in different biological samples [5]. Different software programs used to detect 2DE-gel analysis, in addition to most of the original computer systems, have been developed into commercial packages, such as “PD-Quest, Progenesis, Z3, and mela-nie.” PD-Quest is the most widespread method used in 2DE-gel analysis, and it uses the standard gel analysis workflow already present in the Quest system [85]. Progenesis is also a recent software that provides fully automated gel image processing. It offers more features than PD-Quest by adjusting the link information at various levels to data sets. For example, it can manage the data of 2DE-gel analysis and MS in an integrated manner with different formats [86]. Furthermore, enhancements in high-resolution multistage mass analyzers are used to identify many milk proteins rapidly. These enhancements include linear ion trap, Fourier transform ion cyclotron resonance, and Orbitrap instruments [87]. The Orbitrap mass spectrophotometer has high Se and accuracy. Therefore, it can provide quantitative information for proteins to identify several unknown proteins and peptide sequences and detect the molecular weight of nucleic acids [88].

Chen et al. [89] used a new type of proteomic analysis called an isobaric tag for relative and absolute quantification to screen the differentially expressed proteins in CM caused by an S. aureus infection and identify the most important proteins associated with mastitis. Bacterial [90] and bacterial strains can be determined in a few minutes using MALDI-TOF with Sp and Se values of up to 100% [91].

Proteomic methods can analyze high-throughput proteins, detect and specify them, and predict the changes in protein levels, resulting in a comprehensive understanding of the occurrence of mastitis and the progression of the disease [92]. On the other hand, all progress in proteomics and these technological techniques are expensive and not generally used for routine mastitis diagnosis in farm animals [44]. In addition, they have limitations in their validation procedures because of the lack of species-specific antibodies. Furthermore, there is no distinctive protocol for analyzing many less abundant milk proteins representing 5% of the total milk proteins in the whey or the milk fat globule membrane [93].

Detection of biomarkers

As described before, SCC is the main marker that is dramatically increased in milk samples after inflammation of the udder. Other diagnostic biomarkers for mastitis are available.

APPs

APP has become an important diagnostic indicator used in human and animal medicine. Haptoglobin (Hp) is one of the most abundant APPs produced by the liver, and its concentration is increased in serum because of bacterial inflammation and infection. In addition, it is used to differentiate between SCM and CM and between healthy and diseased cows. Hp is significantly elevated in milk during mastitis [94]. Georgieva et al. [95] suggested that the concentration of plasma Hp in the high SCC group (SCM) was approximately six times higher than that in the normal SCC group.

Serum amyloid A (SAA), which is synthesized in the liver or extrahepatic origins as in the mammary gland, is another pattern of APP. SAA is increased 1,000 times within 24 h of tissue injury because this protein has pro-inflammatory and anti-inflammatory effects. Hence, it can be used to monitor the health status and prognosis of veterinary medicine therapy. The level of this protein is higher in mastitic milk than in normal milk, followed by an increase in SCC. In addition, it is higher in the inflamed quarter than in the normal quarter of the same udder [96]. Eckersall et al. [97] reported a significant correlation between Hp in milk and serum. On the other hand, there was no relationship between the concentration of SAA in milk and serum. In addition, they suggested that the Sp of Hp and SAA is 100%, and the Se of both proteins is 86% and 93%, respectively [97]. Therefore, these proteins can be used to diagnose mastitis. Some researchers used milk amyloid acute phase protein (MAA) to detect SCM [98] and CM [99] because the mammary gland is the primary origin of MAA production in response to an infection. Its level increases rapidly in serum and milk after the mammary gland is infected with different bacteria. SAA is increased under stress conditions. Hence, direct MAA measurements from milk samples are more useful in accurately detecting and evaluating the severity of mastitis [99].

Bovine serum albumin (BSA) is the first protein measured in milk using the radial immunodiffusion method as an inflammatory indicator [100]. Bakken and Thorburn assessed the seriousness and stability of SCM by measuring the concentration of the quarter milk BSA. Milk samples were collected from 51 cows examined twice, with a change in diagnoses from the first to the second examination. The BSA concentration increased as the scores increased on the CMT. The concentration of BSA was higher when bacteria were isolated compared to negative bacteriological findings. In addition, it was higher when “major pathogens,” such as S. aureus or Str dysgalactiae, rather than “minor pathogens,” were isolated [101].

Cathelicidins are polypeptide components of the innate immune system, and as inflammatory proteins, the levels increase in cases of mastitis before the onset of the disease [5]. Addis et al. [102] evaluated the accuracy and Sp of cathelicidins by enzyme-linked immunosorbent assay (ELISA) and reported a positive relationship between cathelicidins and high SCCs. Recently, the functions of cathelicidins in the pathogenesis of S. aureus mastitis were explored in mice. Interestingly, endogenous murine cathelicidin controlled the S. aureus mammary infection, which was confirmed using cathelicidin knockout mice. The intramammary challenge of these mice with S. aureus revealed more intense mastitis with a high bacterial load compared to normal mice. In addition, using an external source of synthetic human cathelicidin or murine cathelicidin resulted in more resistance to S. aureus infection of the murine mammary epithelium by reducing the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6 cytokines [103].

Enzymes

The activity of several enzymes linked to SCM, such as NAGase, LDH, alkaline phosphatase, and arginase, was measured. The increases in NAGase and LDH enzymes are accompanied by tissue destruction. LDH is a sensitive indicator of mastitis because it is released in milk directly from ruptured mammary epithelial cells and phagocytes. Its level in mastitic samples is 63 times higher than in healthy samples. In the case of mastitis, its concentration ranges from 227.07 to 1,524.04 U/L. On the other hand, its level in normal milk ranges between 14.0 and 485.94 U/L [104]. Babaei et al. [105], however, recorded that with the increased percentage of this enzyme in mastitis, LDH is not sensitive for diagnosing mastitis because this increase might be related to an increase in the blood and its transfer to milk through damage to the blood–milk barrier. Nevertheless, Batavani et al. [106] estimated that the percentage of LDH increased more in milk samples than in blood samples, showing that the origin of this enzyme is milk and not blood. Moreover, a previous study [107] suggested that the Se and Sp of LDH were more than 80% and 99%, respectively, in the early diagnosis of mastitis before CM occurred within three to five days.

NAGase is an intracellular lysosomal enzyme released into the milk compartment in response to inflamed cell lysis and after the phagocytosis of neutrophils [26]. Its activity is related to the degree of inflammation. Therefore, it is significantly higher in cases of mastitis caused by major pathogens than in mastitis caused by minor organisms [108]. Chagund et al. [109] revealed a direct relationship between NAGase and SCC. On the other hand, there is a stronger relationship between LDH and SCC. In addition, the activity of the arginase enzyme was higher in milk during udder inflammation. Kandemir et al. [110] stated that the arginase activity of mastitic milk samples exceeds that of normal samples 2.5 fold.

Lactose

Lactose is a milk component that can be used as an indicator for mastitis diagnosis; its concentration decreases as the inflammation associated with mastitis increases. During mastitis, tissue damage leads to a decrease in the activity of secretory cells, so the level of lactose is reduced. The mammary gland is the main organ responsible for lactose production. On the other hand, it can be detected in the blood and urine of animals accompanied by mastitis. Lactose leaks through the blood–milk barrier from the milk to the blood during mastitis [111].

Pro-inflammatory cytokines

Pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-8, IL-6, and CD14 antigen, are defined as fundamental cell surface proteins that facilitate microbial cell wall composition detection by RT-PCR. These cytokines are naturally upregulated by bacterial infections of the udder tissue [112]. TNF-α is a pro-inflammatory cytokine that is increased during inflammation of the mammary gland; its level is increased in coliform- and E. coli-induced mastitis within hours after intravenous injection of lipopolysaccharide in cattle [112]. IL-8 is also one of the most critical chemokines and plays an essential role in mastitis because it induces the migration of neutrophils to the epithelial cells of the mammary gland. Kawecka-Grochocka et al. [112] investigated the levels of RNA expression and the protein concentration of cytokines in dairy cattle udder tissues by RT-qPCR and ELISA, respectively. They reported no alterations in the mRNA levels of TNF-α, IL-18, CCR1, IL-1β, CCL2, IL-8, and IL-6, and the protein concentrations of TNF-α, IL-18, CCR1, and IL-1β in cows with chronic mastitis caused by a staphylococcal infection compared with healthy cows. More importantly, increased IL-8 and CXCL5 concentrations and decreased IL-6 concentrations were observed, suggesting that a chronic mammary gland infection is characterized by decreasing pro-inflammatory cytokines. Vitenberga-Verza et al. [113] reported that immunoreactivity was more pronounced for IL-4, IL-6, IL-12, IL-13, IL-17A, TNF-α, and IFN-γ in mastitis cases.

Advanced instrument detection techniques

Novel techniques for detecting early udder inflammation within hours after infections are under development. For example, transrectal color Doppler sonography is a noninvasive technique that detects the blood flow volume that passes to the mammary gland within a few hours after the infusion of a microorganism. Considering that the amount of blood flow passing to the udder changes after infection, the disease can be detected in the early stages before severe losses occur [114]. B-mode ultrasonography was also used to determine the disturbance in milk secretion and the structural changes in the teat cistern and udder tissue [115]. For the first time in mastitis, Risvanli et al. [116] evaluated the supramammary lymph nodes of cattle by color Doppler ultrasonography because mastitis causes lymphocytic proliferation that leads to morphological changes in the supramammary lymph nodes [117].

Infrared thermography (IRT) is a noninvasive, rapid, safe, and sensitive method to detect the early inflammatory changes accompanied by SCM [118] by measuring the changes in the teat skin temperature. IRT is sufficient alone to diagnose mastitis and detect any changes that have not yet been observed in cows [119]. The Se and Sp of IRT for the diagnosis of SCM are similar to those of CMT [120]. Pezeshki et al. [121] reported a 2–3°C increase in udder temperature in cows infected with E. coli compared to the control. This result is similar to Metzner et al. [122], who reported that the temperature of the dairy cow udder was 2.06°C higher in cases of acute mastitis caused by E. coli infusion than in normal cows. Holstein Friesian cows infected with SCM showed increases in udder temperature of 2–3°C [13], 0.72–1.05°C [123], or 1.35°C on the surface of the udder between infected and healthy quarters [124].

Mastitis detection during automatic milking

Robotic milking has been used in recent years and affords an ideal arrangement for checking mastitis online [4]. Therefore, reliable and sensitive methods are required (Fig. 4). The automatic milking system has become a standard approach in large farms, and it affects the welfare and health of animals by reducing the percentage of infection [125]. Any online mastitis diagnosis is achieved by assessing the SCCs, EC, or color. Sensors as an automated online CMT allow more accessible and widespread screening of high SCC cattle within a herd, making it accurate for identifying mastitic animals [126]. EC is the most commonly used online test. Nevertheless, any changes in conductivity may be a valuable indicator, so it is not a consistent or sensitive parameter for a definite diagnosis [127]. The milk color is another indicator used in automatic programmed milking systems for mastitis infection by observing a yellow color or fresh blood in the milk. The milk fat content influences the milk color, so some color sensors cannot detect SCM [5]. Therefore, the goal of several recent research determinations is the progress of new sensors with higher sensitivities. For example, the electronic tongue is a chemical array sensor that can measure sodium, potassium, and chloride ions, which increase during mastitis along with (organic and inorganic) anions and cations [128] (Fig. 4). In addition, the electronic nose is a gas-array-based sensor involved in the binding of many gas sensors with volatile substances, such as sulfides, amines, ketones, and acids (Fig. 4), to distinguish infected from healthy milk [129]. More recently, the patterns of volatile metabolites can be detected to recognize several microorganisms, such as S. aureus, CNS, E. coli, and Streptococci [130,131] (Fig. 4). Furthermore, the lactate level can also be considered a primary indicator for mastitis detection. An increased lactate concentration is directly related to metabolic activity owing to the limited amount of available oxygen in the glands [128]. A lactate screen-printed sensor that covers the lactate oxidase produced on the sensor surface has been developed. The sensitivities of these sensors are higher than other sensors, but their ability to identify subclinical cases is unclear. Another study developed a sensitive paper-based colorimetric device for rapidly determining SCC, which can be used for diagnosing mastitis [132].

Fig. 4. Current and potential “online” analysis for mastitis detection in milk samples. An automatic milking system is becoming a common approach in large farms by using color, EC, and SCC sensors as “online” assays. On the other hand, sensors for detecting NAGase, haptoglobin, and gases produced by bacteria have yet to be incorporated ‘online.’ These sensors show great potential for the accurate detection of mastitis.

APP, acute phase protein; CMT, California mastitis test; TCDS, transrectal color Doppler sonography; IRT, infrared thermography.

Furthermore, biosensors are becoming the next generation for exploring basic fields [128,132]. Therefore, the detection of mastitis is improved by developing biosensors using a biological receptor, such as an enzyme, antibody, or nucleic acid, combined with a transducer to yield a related signal and observe a specific biological action, such as antigen-antibody reactions to differentiate between SCM and normal milk [128] (Fig. 4).

Biochips for mastitis detection

New trends towards mastitis diagnosis using microfluidics or biochips have been applied. Disposable microchips with a portable reader can be used for SCC measurements in milk samples by mixing the sample with a solution that can destroy the somatic cells and expose it to a fluorescent dye to stain the DNA. The sample is then exposed to the microchip, and the fluorescence is measured with a portable reader system [133]. Portable platforms are used for bacterial cell isolation, identification, and counting by binding the cells with magnetic materials. The magnetically labeled cells can be detected by magnetoresistive sensors integrated with the microfluidic channels [134]. Similarly, another disposable method involves mixing the samples with a metachromatic detergent to stain the white blood cells and identify them under a fluorescence microscope [135].

Nanotechnology

Nanotechnology provides a new opportunity for the progress of sensitive, fast, specific, and cost-effective approaches for diagnosing bacterial infections, including mastitis [136]. Nanotechnology can grasp and discriminate target molecules or microbes from other constituents in a complex sample matrix [137]. These molecules are attached to affinity probes, such as antibodies and nucleic acids, that can distinguish between microbe biomarkers. Magnetic, gold, and fluorescent nanoforms are examples of nanomaterials used for bacteriological analysis [138]. Nanoparticles can be designed to be fluoresced by light motivation or two-photon excitation [138]. The magnetic detection technique is characterized by the Se, simplicity, flexibility, speed, and cost-effectiveness [139]. Nevertheless, false-positive results may be produced when using magnetic detection methods due to nanoparticle agglomeration caused by the heterogeneity and EC of unsound milk. This may be avoided using detergents, such as Tween 20, which increases nanoparticle mobilization, bacterial cell distribution, and the homogeneity of the milk matrix [140]. Moreover, a false-negative result may be produced because of variations between the nanoparticles and antibodies or a failure in the magnetic labeling of bacterial cells [141]; in addition, milk clotting leads to an obstruction of the microchannel [140] and signal cancellation [142].

DISCUSSION

The continuous monitoring of mastitis can be successful by detecting the inflammation in the early stages of the disease and consequently detecting and managing SCM infections. In the future, the diagnosis of mastitis should be more sensitive, fast, specific, and cost-effective. Therefore, this review summarized the merits and demerits of the most traditional and advanced methods used in a mastitis diagnosis to choose the best tools (Table 2). Bacteriological culturing is still considered the gold standard in identifying pathogens worldwide. Molecular diagnostic techniques have also become more common and reliable tools for identifying pathogens causing mastitis; they can be used as a confirmative method in addition to BCs to obtain more sensitive and accurate results. Furthermore, some authors are using complementary tools to confirm the results, e.g., evaluating APPs as MAAs and some enzymes, such as LDH and NAGase, as biomarkers for the inflammatory changes accompanied by mastitis in addition to measuring the udder skin temperature by IRT. over the last decade, researchers have shifted their opinions on using microfluidic biochips, biosensors, nanotechnology, and automatic milking systems. Nevertheless, the number of farms and herds is growing rapidly. Therefore, further research is needed to improve the ability to overcome any limitations and increase the success of these techniques in early diagnosis. Eventually, genomics and proteomics approaches for state-of-the-art biologics against bovine mastitis infectious agents will be highlighted, which will pave the way for the settlement of the most effective platforms to control bovine mastitis.

Table 2. Advantages and disadvantages of the techniques used in mastitis diagnosis.

			Methods		Advantages	Disadvantages	References
i. Identification of pathogens causing mastitis
	1		Phenotypic method (BC)		• Available for most laboratories.	• Bacterial growth is inhibited in the presence of preservatives and antibiotic residues, if inappropriate media are used for the microorganisms, and through “intermittent shedding.”	[94,95]
					• Broad spectrum screening capacity.	• Time-consuming and laborious.
					• BC reflects true IMI through the multiplication of microorganisms.	• Do not detect the degree of inflammation related to the infection.
					• The growth cultures are still suitable for further repetition or confirmation.	• Not suitable for the isolation of some pathogens, such as Mycoplasma.
						• Serotyping is not efficient enough.
						• Public health hazard from biological contamination during pathogen enrichment.
	2		Genotypic method (PCR methods)		• Sensitive, specific, rapid.	• Using limited access in developing countries.	[53,163,169]
					• Storage of PCR products for a long time in a freezer.	• Need a well-trained person.
					• Differentiate between clonal outbreaks and multiple strains.	• Using ethidium bromide (carcinogenic substance) in conventional PCR.
					• Handling of PCR products is not required in RT-PCR.	• Improper extraction of DNA from milk samples due to the presence of PCR inhibitors in milk.
					• Detection and quantification of more types of pathogens by multiplex PCR.
					• MicroRNA used for the early detection of infection acts as a good marker for mastitis detection; this parameter is not affected by “acidity, temperature, freezing, thawing and RNAse enzyme.”
					• Different strains from different pathogens in the same sample can be detected by a combination of PCR and Microarray.
	3		Mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS)		• Detection of bacterial strains and bacterial species within a few minutes.	• Too expensive for use in diagnostic laboratories.	[164,165]
					• Reliable and easy to perform.	• It is limited to identifying an existing database of specific protein profiles.
					• Sensitivity and specificity up to 100%
	4		Magnetic detection methods depend on nanoparticle administration		• Sensitive, simple, rapid, and cost-effective.	• Agglomeration of nanoparticles.	[170,171,172]
					• Flexible, whereas more than antibodies can be used to identify different pathogens causing mastitis.	• Failure of magnetic labeling of bacterial cells.
						• Signal cancellation.
						• Microchannel obstruction by clotted milk.
						• It is not used on a large scale.
ii. Detection of mastitis markers
	1		Cell counting
		1.1		SCC	• Used worldwide for decades for screening the IMI and evaluating the effectiveness of a mastitis control program by directly measuring the number of somatic cells in milk.	• It is affected by some factors such as age, season, stress parturition, and lactating cycle.	[15,96,173]
						• Does not correlate with udder infections.
		1.2		CMT	• Simple, inexpensive, and rapid method used for the indirect count to SCC.	• It is not used for a large-scale monitoring purpose.	[26]
					• It is the major cow site test used to detect the infected quarter after the bacteriological examination.	• Its sensitivity decreased with the decrease of SCC.
						• Affected by season, stress, and parturition.
						• Its interpretation differs between samples.
		1.3		Wisconsin mastitis test	• Measuring the somatic cell indirectly depends on measuring the height of the gel produced in the tube.		[44]
	2		EC		• Measurement of anions and cations in milk online.	• Low specificity and influenced by some factors such as age, breed, lactation stage of the cow, temperature, and PH of the milk.	[38]
	3		APPs
		3.1		Haptoglobin	• Differentiate between clinical and subclinical mastitis and between healthy and diseased cows.		[94]
		3.2		Serum and milk amyloid A	• Its level is not detected in the milk of healthy animals, so it is unaffected by factors other than mastitis. Therefore, it acts as a good indicator for a mastitis diagnosis and monitoring the health status and the prognosis of the therapy.	• SAA is affected by factors other than mastitis, such as stress, so MAA is more of an indicator than SAA.	[174,175,176]
					• MAA directly from milk samples is more useful in accurately detecting and evaluating the mastitis severity.
		3.3		Ceruloplasmin	• Good indicator in the early diagnosis of clinical mastitis.	• Less common than other acute-phase proteins.	[177]
		3.4		CRP	• Its concentration increased in the milk of mastitis cow.	• There is no correlation between CRP and SCC, so it is not the choice for mastitis diagnosis.	[178]
		3.5		Cathelicidins	• Its concentration increased in cases of mastitis before the onset of the disease.		[179]
					• Assessing the severity of mastitis.
	4		Enzymes related to the inflammations
		4.1		LDH	• It is used in the early diagnosis of mastitis before the clinical mastitis occurs within three to five days.		[107]
					• Its sensitivity and specificity are more than 80% and 99%, respectively.
					• There is a strong relationship between LDH and SCC.
		4.2		NAGase	• Detect the degree of inflammation during mastitis.		[109]
					• There is a direct relationship between NAGase and SCC.
		4.3		Arginase	• It is increased by 2.5 times during subclinical mastitis than in normal cases.		[110]
	5		Pro-inflammatory cytokines such as TNF-α and IL-8		• These chemokines increased with the increase of inflammation associated with mastitis.		[112]
					• IL-8 is the most common cytokine responsible for migrating neutrophils to udder epithelial cells.
	6		Transrectal color Doppler sonography		• Detect the disease in the early stages before the occurrence of severe losses depending on the detection of blood flow volume to the mammary gland after being infused with the pathogen within hours.		[114]
	7		IRT		• Noninvasive, rapid, safe, and sensitive method.	• It may be affected by weather conditions, dirt, sunlight, and moisture.	[116]
					• Used to detect early inflammatory changes that accompanied subclinical mastitis.
					• IRT is sufficient to be used alone in the diagnosis of mastitis and can detect any changes that have not yet been observed in cows.
	8		Histopathological evaluation		• It precisely gives information on the alveolar structure and shows the state of subsequent secretory cell differentiation.	• Special precautions are needed to obtain  biopsies from live animals.	[180]
						• It requires special types of stains.
						• A professional technician is needed for sample preparation and interpretation of results.
	9		Immunoassays, e.g., ELISA		• High sensitivity and specificity.	• Temporary readouts: detection is based on enzyme/substrate reactions, and readouts must be obtained quickly.	[181]
					• Easy and rapid to perform.
					• Simplicity of reading results.
	10		Biochips		• More than seven pathogens causing mastitis could be detected in one reaction by developing multiplex biochips.		[46]
					• Accurate, rapid, and pathogens causing mastitis could be detected in a single reaction by developing multiplex biochips.

BC, bacteriological culture; IMI, intramammary infection; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; SCC, somatic cell count; CMT, California mastitis test; EC, electrical conductivity; APP, acute phase protein; MAA, milk amyloid acute; SAA, serum amyloid A; CRP, C-reactive protein; LDH, lactate dehydrogenase enzyme; NAGase, N-acetyl-β-D-glucosaminidase; TNF, tumor necrosis factor; IL, interleukin; IRT, infrared thermography; ELISA, enzyme-linked immunosorbent assay.

Molecular techniques are essential for detecting pathogens and predicting the probability of tissue infections in the future. Therefore, the methodology used for DNA extraction from milk samples is important because milk contains protein, fat, and calcium, which act as inhibitors for PCR [143] and may affect the accuracy of bacterial species detection [144]. Furthermore, samples from diseased cows contain other PCR inhibitors, such as cellular debris. Lima et al. [145] used a new method to isolate DNA using the amplicon sequencing technique, which is based on exposing samples in the cell lysis step to a higher temperature of 70°C for 10 min, followed by a bead beating process for 15 min. In addition to the washing step, milk samples are subjected to a cleaning process to enhance the transparency of the final DNA template.

Furthermore, the amplification of target DNA sequences using different PCR methods is a recently developed method. For example, recombinase polymerase amplification (RPA) uses 76 other proteins in addition to polymerase recombinase proteins and single-strand binding proteins. The isothermal condition is very important for the reaction. Many alternatives to RPA, such as reverse transcription-recombinase polymerase amplification (RT-RPA), multiplex RPA, and on-chip RPA, are also accessible [2]. A unique diagnostic method called loop-mediated isothermal amplification was established to detect the presence of Str. agalactiae [146], Str. uberis [147], and S. aureus [148] in milk.

In addition to the development of molecular biology methods, novel indicators specific to mastitis are needed. Fourier transform infrared spectroscopy is a technique used to differentiate between closely related bacterial species [149] based on the DNA fingerprinting of each organism. Therefore, in the future, this technique may be developed for diagnosing mastitis. Future pen-side mastitis tests should consider the need to detect cows susceptible to new or existing intramammary infections before calving while being economical and user-friendly at the same time. Developing better, faster, cheaper, and more convenient tests will encourage appropriate interventions to prevent transmission, reduce antimicrobial resistance, and minimize financial losses.