The name “mastitis” comes from the Greek word “matos,” which means “breast” or “udder,” and the suffix “itis,” which means “inflammation”
(Sangam et al., 2021; Ibrahim, 2017). Bovine mastitis is an inflammation of the parenchyma of the mammary gland that can cause pathological changes in the glandular tissue as well as physical, chemical and pathogenic bacteria overload in milk
(Mellata, 2022). In clinical instances there may be discoloration, a clot, a high number of leucocytes in milk and swelling, pain, heat and edema in the udder
(Thakur et al., 2020). Bubaline mastitis is less common and economically significant disease
(Johri et al., 2023; Jhambh et al., 2017). Mastitis in cattle and buffalo is a major global health issue that affects countries such as India, Canada, Germany, the United Kingdom, the Netherlands and the United States of America
(Sharun et al., 2021). The daily loss of milk from bovine mastitis ranged from 1.0 to 2.5 kg in the first two weeks and the overall loss over the course of the lactation was 110 to 552 kg, depending on the parity and the time of commencement. Since cows will not achieve their peak milk supply throughout the remaining portion of lactation, mastitis also has a long-lasting influence on milk yield
(Rajala-Schultz et al., 1999). India is the world’s largest producer of milk (both cow and buffalo milk together). In India, mastitis causes a 21% decrease in milk production and an estimated Rs. 575 million in economic losses annually
(Bardhan et al., 2013). Mastitis milk poses a zoonotic risk and is not fit for consumption or sale, which results in significant financial losses. Animals with infected udders are less expensive to buy and place a financial strain on their owners due to medical expenses
(Seegers et al., 2003). The diagnosis of mastitis is the main requirement for the dairy business to provide clean milk for reasons of economics, public health and animal welfare. For the goal of managing or treating mastitis, early recognition of the condition is crucial for prompt, precise and timely diagnosis. This calls for the use of both traditional and cutting-edge diagnostic procedures. Conventional procedures are generally non-specific, but they are also straightforward to use, inexpensive and quickly available in the field. The advanced tests are expensive, necessitate technical expertise and require sophisticated facilities and infrastructure; yet, they are typically accurate and specific for various kinds of mastitis
(Chakraborty et al., 2019). This review article describes the advance diagnostic methods for udder infection.
Major pathogen causing mastitis
Mastitis may result from any type of physiological harm, pathogenic germs, or chemical irritants that elicit an immunological response
(Bramely et al., 1996). Bacteria, fungi and yeasts may play a role; but of these, bacteria have by far the largest part
(Kibebew, 2017). Pathogenic bacteria and mastitis were found to be associated in 1887; however, the primary pathogens were not recognized until the 1940s. More research on the etiology of bovine mastitis was made possible by the 1960s discovery of its multifactorial nature
(Ndlela et al., 2016). It has been claimed that around 140 distinct pathogenic species exist
(Zeryehun et al., 2017). It is thought that the primary cause of mastitis is bacterial intra-mammary infection (IMI). Both Gram-positive and Gram-negative bacteria can cause mastitis
(Ashraf and Imran, 2020). Bovine mammary gland infections can be caused by bacteria belonging to three main categories: opportunistic, environmental and contagious pathogens
(Hawari and Hassawi, 2008). During the milking process, infectious bacteria that reside on the udder spread from infected to uninfected teats.
Staphylococcus aureus,
Mycoplasma bovis and
Streptococcus agalactiae are the principal ones. Opportunistic infections are able to infiltrate the gland’s inner lining due to their potent adhesive qualities. They may result in sporadic bouts of severe mastitis
(Fox and Gay, 1993). Environmental pathogens often live in the bedding and housing and they often enter the teat canal when the cow is being milked. Environmental mastitis is primarily caused by
E.
coli (Gunther et al., 2011). Mastitis is typically caused by three common streptococcal species:
S.
uberis,
S.
dysgalactiae and S.
agalactiae. Other environmental bacteria include
Serratia,
Pseudomonas,
Proteus and other Gram-negative bacteria as well as
Klebsiella,
Citrobacter and
Enterobacter spp, including
E.
faecalis and
E.
faecium (Radostitis
et al., 2000). Opportunistic pathogens known as coagulase-negative staphylococci (
S.
epidermidis,
S.
simulans,
S.
saprophyticus and S.
chromogenes) infect the lining of the teat or udder surface
(dos Santos Nascimento et al., 2005). The two most common pathogens worldwide that cause bovine mastitis are coagulase-negative staphylococci and S. aureus
(Graber et al., 2007). Candida spp.,
Cryptococcus neojormans,
Saccharomyces spp. and
Torulopsis spp. are instances of yeast infections, while Aspergillus jumigatus,
Aspergillus nidulans,
Pichia spp.
and Trichosporon spp. are examples of fungal infections. Leptospirosis infections, such as
Leptospira interrogans serovar
pomona and particularly
Leptospira interrogans hardjo, damage the blood vessels in the mammary gland and result in a noticeable irregularity in the milk. Algal infections of mammary gland include
Prototheca trispora and
Prototheca zopfii. Although they are not very important, several viruses can also cause mastitis in cattle. Some other bacteria which involve in mastitis are
Citrobacter spp.,
Enterococcus jaecalis,
Enterococcus jaecium,
Proteus spp.,
P.
aeruginosa and Serratia spp.; other Gram-negative bacteria include
Nocardia asteroides,
Nocardia brasiliensis and Nocardiajarcinica; Histophilus somni; Pasteurella multocida,
Pasteurella mannheimia;
Campylobacter jejuni;
B.
circus. Anaerobic bacteria have been identified from mastitis instances; these bacteria are typically found in conjunction with other facultative bacteria, such as
Fusobacterium necrophorum,
Eubacterium combesii,
Prevotella melaninogenica and Peptostreptococcus indolicus (Hamadani
et al., 2013).
Types of mastitis
Based on the level of inflammation, mastitis is categorized into three classes: clinical, sub-clinical and chronic mastitis
(Han, 2020). Visible abnormalities such as a reddened and swollen udder make clinical mastitis clear and easy to recognize; fever milk appears watery and contains flakes and clots
(Maros Cobirka, 2020). Acute, sub-acute and per-acute forms of clinical mastitis can be distinguished based on the level of inflammation.In sub-clinical mastitis, there are no outward signs of abnormalities in the udder or milk; yet, as the somatic cell count (SCC) rises, milk production falls
(Kibebew, 2017). So the development of a rapid diagnostic test for mastitis is necessary, especially for subclinical mastitis in milk at the earliest stage of infection
(Salvador et al., 2014). The first pathogenic change observed in subclinical mastitis is the presence of leukocytes and erythrocytes into milk, which increases the permeability of mammary capillaries and triggers an inflammatory response
(Tripti et al., 2018). Subclinical mastitis was more common in India and Staphylococcus species were more common than other pathogens in mastitis
(Krishnamoorthy et al., 2021). Additionally, it has been documented that environmental pathogens are more likely to induce the clinical form of mastitis, while infectious causal pathogens are more likely to generate long-lasting subclinical mastitis infections, which are thought to be persistent in a significant number of animals
(Sangam et al., 2021). When an animal receives insufficient care during the clinical stage of mastitis, chronic mastitis develops.In case of chronic type mastitis, quarters grow hard and antibiotic treatment frequently fails.The structure of milk is often lumpy and the udder swells up extremely red
(Sangam et al., 2021).
Epidemiology
The global prevalence of clinical mastitis (CM) and subclinical mastitis (SCM) was 15% and 42%, respectively
(Krishnamoorthy et al., 2021). Sub-clinical mastitis is 15-40 times more common than clinical mastitis
(Ndahetuye et al., 2019).Cattle had a greater rate of clinical mastitis (20%) than buffaloes (11%), whereas buffaloes had a higher rate of subclinical mastitis (66%) than cattle (53%). Mastitis prevalence was 85.3% in cows and 78.1% in buffaloes, according to a retrospective study conducted on the antibiogram and prevalence of mastitis in Eastern Haryana’s cattle and buffaloes over a six-year period from 2004 to 2009
(Bhanot et al., 2012). In addition, both species’ month-wise prevalence was higher in warm, muggy months
(Ali et al., 2021). The economic loss due to subclinical mastitis higher that the acute mastitis in buffaloes
(Kashyap et al., 2019). The incidence was higher in the hindquarters of buffaloes than cows and both species’ right hindquarters were shown to be more vulnerable. The incidence of forequarters was found to be higher in cows than in buffaloes, with the right forequarters being more susceptible
(Swami et al., 2017). Compared to cows, buffaloes are thought to be less prone to mastitis. However, the viruses that cause the infection can spread swiftly once the buffaloes are infected since buffalo milk is high in nutrients. Tighter teat sphincters in buffaloes help to better prevent pathogen invasion and the mucin
-1 (MUC1) gene, which primarily shields the cell surface from environmental pathogens. This is one of the reasons why buffalo mastitis is less common than bovine mastitis
(Amin et al., 2023; da Rosa et al., 2020). Thus, it was discovered that the incidence was higher in the hindquarters of buffaloes than cows and that both species’ right hindquarters were more vulnerable
(Swami et al., 2017). The hind quarters are more vulnerable to environmental and fecal pollution because of their anatomical location, high production capacity and greater pendulousness than the front parts
(Kashyap et al., 2019; Shukla et al., 2016). It was discovered that right side quarters were more vulnerable. Because the animals assume a sitting position on their right side, the pressure on their right side quarters widens the teat canal, allowing pathogenic organisms to enter
(Swami et al., 2017; Shukla et al., 2016). The milkman’s initial handling of the animal’s right rear quarter while milking may have increased the risk of infection (Fig 1)
(Shukla et al., 2016).
Risk factors
Mastitis is considered to be a typical example of complex diseases, known to be established as a result of the interactions of three bio-systems namely the causative agent (pathogen), the animal (host) and the environment in which the animal lives.
Pathogen factor
The pathogen factor classified into 2 types based on the bacterial origin-contagious and environmental
(Lakew et al., 2019).The term “contagious mastitis” describes mastitis that can spread from cow to cow, particularly when milking
(Breen 2017). Environmental infections typically reside in the herd’s bedding and housing rather than on the udder and teat skin of cows. The best way to characterize them is as opportunistic pathogens, as they hunt for opportunities to infect
(Bradley et al., 2012).
Host factor
Consists of animal age, breed, number of parities, stage of lactation, the shape of udders, teat end morphology, teat and udder lesion
(Amin et al., 2023).
Age
Higher in buffalo aged 7 to 18yr than in those aged 3 to 6 yr due to the structural changes in the udder and teats and the gradual suppression of the buffalo immune system. Older cows are more susceptible to infections, most probably because of the wider or permanently partially-open teat canal as a result of frequent milking
(Kibebew, 2017).
Breed
Relevance of bubaline mastitis is higher in crossbred buffalo than in indigenous breed because crossbred buffaloes can produce more milk
(Amin et al., 2023).
Stage of lactation
Prevalence of bubaline mastitis in the early lactation stage (14 to 100 days) is higher than in the late lactation stage (more than 200 days) and the mid-lactation stage (100 to 200 days) due to a gradual increase in milk production
(Kavitha et al., 2009).
Number of parity
Multiparous cows are more vulnerable to IMI than primiparous cows due to immune-incompetence
(Elbayoumy et al., 2024; Jingar et al., 2014).
Udder structure
The prevalence of bubaline mastitis is higher in buffaloes with bowl- or round-shaped udders than cup shape udders and higher in cylindrical and round teat ends than pointed teat ends
(Amin et al., 2023).
Enviromental
FactorAnimal welfare and health are significantly impacted by the management techniques used by the herds and the surrounding environment. Mastitis occurrence and severity can be decreased by maintaining a clean, pleasant herd. Mastitis can occur more frequently in cows due to factors such as excessive stocking density, polluted floors, damp bedding, inadequate ventilation and hot, humid weather that encourage the growth of mastitis bacteria and increase cow exposure
(Sharun et al., 2021).
Advance diagnostic methods
Mastitis is a complex illness that affects dairy cows all over the world. It has a big impact on a country’s economy and social cohesion. Therefore, it is imperative to come up with a prompt and accurate solution
(Said et al., 2022).Advanced testing for mastitis are quantitative, highly specific and sensitive, while conventional tests are typically qualitative with lower specificity and sensitivity
(Chakraborty et al., 2019; Godden et al., 2017). The latest developments in the diagnosis of mastitis are listed in Fig 2.
Proteomic-based diagnosis
By this method pathogens could be accurately, sensitively and quickly
diagnosed(Kour et al., 2023). The term “proteome” refers to all of the proteins that are present in a cell or tissue at any given time. The goal of proteomics research is to quantify changes in protein abundance throughout the pathological circumstances being studied, as well as to identify the proteins that are present in tissue samples during different physiological stages
(Katsafadou et al., 2019). Proteomic investigations of milk obtained during bovine mastitis have been conducted using a variety of techniques, including tandem mass spectrometry and liquid chromatography combined with MALDI-TOF MS after two-dimensional gel electrophoresis (2D-GE)
(Chakraborty et al., 2019). Barreiro et al., identified S.
aureus,
E.
coli,
Strep.
agalactiae,
Strep.
dysgalactiae and Strep.
uberis from contaminated milk samples with use of MALDI-TOF MS
(Kour et al., 2023). Early on in subclinical mastitis, milk whey contains protein indicators that can be used as a reliable diagnostic tool to identify mastitis using comparative proteomics
(Bian et al., 2014).
Sensors for mastitis detection
These diagnostic tools often cause the animal as little stress as possible when detecting mastitis. In large farms, these diagnostic systems are quite useful.Sensor systems are employed in the detection of clinical mastitis in cows
(Hogeveen et al., 2010).Manual milking has been superseded by machine or automatic milking in large, well-organized dairy farms. The automatic detection of mastitis cases from such a large number of animals requires the use of suitable sensing technology, such as quarter-based milk electrical conductivity (EC) and in-line monitoring of somatic cell count (ISCC) sensing technique
(Kamphuis et al., 2008).
Enzymatic biomarker
There is release of various enzymes in the milk due to the immune responses of the animals against various infectious. There is a tendency of reduction of the enzymes that deal with synthesis of milk along with increased activity of the enzymes found in relation to inflammation
(Chakraborty et al., 2019). Increase in the activities of enzymes that originate from phagocytes such as N-acetyl-D-glucosaminidase (NAGase), milk LDH, ALP, arginase and catalase along with b-glucuronidase
(Preethirani et al., 2015). With its high sensitivity and specificity and ease of measurement, lactate dehydrogenase (LDH) activity in milk has been demonstrated to be a promising biomarker for subclinical mastitis in buffaloes
(Singh et al., 2016).
Nanotechnology based diagnosis
Biosensors are devices with a physical nanotransducer (sensor) and bioreceptors specific to the antigen or chemical under study. These sensors detect the presence of specific biological substances via electrical signals
(Martins et al., 2019). For instance, the development of a real-time mastitis sensing diagnostic based on the identification of acute-phase proteins produced by the liver (such as haptoglobin) was made possible by nanotechnology
(Nirala and Shtenberg, 2020). The development of a colorimetric biosensor assay based on magnetic nanoparticles was prompted by the discovery that plasmin’s increased proteolysis of casein during mastitis signifies a decline in milk quality. The milk from animals with and without mastitis can be distinguished using this assay. A direct indicator of plasmin proteolytic activity is the biosensor’s heightened golden color, which is caused by plasmin bound to magnetic nanoparticles being present as a monolayer across its surface. The in vitro levels of plasmin (1 ng/ml) in the milk samples can be detected by this biosensor with extreme sensitivity
(Chinnappan et al., 2017).
Infra-red thermography (IRT)
The ability of both CMT and infrared thermography (IRT) to differentiate between clinical and subclinical mastitis makes them practically identical. A novel method for early mastitis diagnosis that is practical, portable and may be applied on-site is infrared thermography. It is predicated on the temperature differential between diseased and healthy udders. The degree of udder infection is ascertained by analyzing the heat images that thermal cameras capture. Warmest region appears as white or red, whereas the coolest region appears as blue or black (Fig 3 and Fig 4)
(Sathiyabarathi et al., 2016).
Specific immunoassays
Immunoassays have been used to identify milk amyloid A, acute phase proteins (haptoglobin) Hp, Cytokines, such as tumor necrosis factors and interleukins. which rise in milk during inflammation, in the diagnosis of bovine mastitis
(Al-Rasheed et al., 2022;
Duarte et al., 2015). A number of research provide sophisticated instruments for the earliest diagnosis, such as an indirect ELISA for the detection of antibodies against
Streptococcus agalactiae rAP1-BP-AP2 proteins and rSip-PGK-FbsA fusion protein and a biomarker-based Liquid Phase-Blocking ELISA for subclinical mastitis
(Kour et al., 2023; Bu et al., 2017). For the purpose of identifying and evaluating the cathelicidin proteins found in the milk of buffalo, researchers have created and standardized ELISA. When mastitis occurs, the milk secretes cathelicidin, which are tiny proteins with antibacterial properties linked to innate immunity
(Puggioni et al., 2020; Chakraborty et al., 2019).
Specific culture
Microbiological/bacteriological culture is utmost required for microbial or bacteriological etiology of (subclinical) mastitis
(Abdelmegid et al., 2018). However, in 10-40% of cases with clinical mastitis at the quarter level, a bacteriological investigation of milk samples reveals no growth. Such a situation could arise for a number of reasons, such as the presence of very few organisms or the possibility that samples contain diseases like Mycoplasma spp. that need to be cultured using specialized media and techniques
(Chakraborty et al., 2019)., Bacterial culture techniques are much less sensitive (32.2%) than PCR assays (70.6%) in identifying the pathogens responsible for mastitis
(Vidic et al., 2018).
Polymerase chain reaction (PCR)
PCR is culture-independent methods for identifying bacterial pathogens in milk. PCR is faster(result within 4 hours) and more sensitive compare with traditional culture
(Adkins and Middleton, 2018). It involves DNA extraction and repeated cycles of denaturation, amplification and replication, in which segments of deoxyribonucleic acid (DNA) are continuously multiplied.A particular type PCR, known as multiplex PCR, can amplify multiple sequences of DNA in one reaction
(Journal et al., 2019). RT-PCR uses reverse transcription to produce a DNA template from an RNA source that can then be amplified
(Lu et al., 2014).Reverse transcription (RT)-PCR tests have been developed to both identify and measure pathogens associated with mastitis in milk. The application of these molecular techniques has grown in popularity for the purpose of differentiating bacterial strains within a species
(Shome et al., 2011). RT-PCR allows for instantaneous detection, can be quantitative (qRT-PCR) and can even identify tiny folds (2-folds) also
(Keane et al., 2013).However, conventional PCR is non-quantitative and provides a basic identification of the organism based on the amplification of genetic material or DNA at the end point. It also has low resolution because it can only detect at higher folds (10 or more)
(Duarte et al., 2015). PCR is asensitive (76.9-100%) and specific (63.3–98.7%) technique for diagnosing mastitis
(Paramasivam et al., 2023). PCR has been applied to both single and large-scale milk samples and for clinical and subclinical mastitis
(Syring et al., 2012).
Microbial typing/finger printing/sequencing/characterization
Sequencing has emerged as a key diagnostic technique for mastitis that allows bacteria to be identified by species, subspecies and strain level
(Chakraborty et al., 2019). Bovine isolates from human isolates must be distinguished from each other due to the zoonotic significance of the isolates’ subspecies differentiation
(El-Sayed et al., 2017). For the purpose of genotyping, a variety of molecular techniques have been used, including ribotyping (
Choudhary, 2018), pulsed-field gel electrophoresis (PFGE)
(Pumipuntu et al., 2019) amplified fragment length polymorphism (AFLP)
(Mohajeri et al., 2016), random amplified polymorphic DNA (RAPD)
(Tomazi et al., 2018) and multilocus sequence typing (MLST)
(Pumipuntu et al., 2019). DNA is utilized in these techniques after being digested with restriction enzymes, amplified using PCR, sequence analysis, or by combining all of these techniques
(Choudhary et al., 2018). Multi-locus variable number tandem repeat analysis (MLVA) and ribotyping, as well as study of transfer DNA intergenic spacer length polymorphism, can be carried out at the strain and species levels respectively
(Pinho et al., 2012). S.
aureus isolates from bovine mammary secretions can be successfully subjected to genetic analysis using PFGE in conjunction with binary interspace (IS) typing. It has been discovered that binary IS typing is a reliable technique that is easy to use and has a lot of potential to develop into a potent instrument for characterizing bacterial strains
(Zadoks et al., 2001). PFGE is used to separate large DNA molecules. Better size resolution can be achieved using PFGE as opposed to agar gel electrophoresis. It has been determined that the PFGE procedure is quite appropriate for studying the
streptococci that cause mastitis in cows
(Santos-Sanches et al., 2015). Both PFGE and MLST have been used to genotype and characterize different strains of
S.
aureus that cause mastitis in cows
(Pumipuntu et al., 2019). For the purpose of differentiating
S.
uberis isolates from milk, MLST was developed. By using this specific technique, two new clonal complexes have been identified: sequence type (ST-86) and ST-143
(Tomita et al., 2008).
Recombinase polymerase amplification (RPA)
It is now possible to amplify specific DNA sequences in a way that differs from PCR thanks to a recently developed and promising technology. The introduction of two additional proteins in addition to the polymerase-recombinase proteins and single-strand binding proteins-is essential to the novel technique known as recombinase polymerase amplification (RPA). Under isothermal conditions, the reaction is conducted. It is possible to utilize the used cycler as a portable device because it is noticeably smaller than PCR thermocyclers. There exist numerous RPA variations, including multiplex RPA, on-chip RPA and reverse transcription recombinase polymerase amplification (RT RPA)
(Daher et al., 2015; Kersting et al., 2014).
Micro RNA (miRNA) based diagnosis
MicroRNAs are naturally occurring, tiny, non-coding RNAs that are produced by different kinds of cells and released into extracellular environments and biofluids, where they control a range of biological activities within cells
(Srikok et al., 2020). Changes in the expression of different microRNAs (MIR146A, MIR155, MIR184, MIR24-3p, MIR148, MIR486 and LET7A-5p) were seen in the in vitro challenge investigations including
E.
coli lipopolysaccharides and
Staphylococcus aureus enterotoxin B. MIR184, MIR24-3p, MIR148, MIR486 and LET7A-5p were identified by
Jin et al., (2014) as distinct microRNAs linked to
E.
coli intramammary infection (IMI)
(Jin et al., 2014).
Highresolution-melt analysis
Using traditional culture-based approaches compromises the accuracy of mastitis pathogen detection. Here, we describe a new, quick assay that uses high-resolution melt analysis (HRMA) of 16S rDNA sequences to screen for the speciation of bacterial mastitis pathogens. A 290 bp amplicon was obtained from the real-time PCR amplification of the 16S rRNA gene segment, which spans the variable regions V5 and V6. Initially, a library comprising the melt curves of nine prevalent pathogens linked to bovine mastitis was created. Three of the isolates-
Arcanobacteriumpyogenes,
Corynebacteriumbovis and
Streptococcus dysgalactiae-were field isolates related to bovine mastitis, while the remaining six-
Escherichia coli,
Streptococcus uberis,
Klebsiellapneumoniae,
Staphylococcus aureus and
Mycoplasma bovis-were type strains. Three of the type strains were identified from illnesses in cows, whereas the other four,
S.
aureus,
K.
pneumoniae,
S.
agalactiae and E.
coli, were discovered to be of human origin.Second, ten bovine mastitis field isolates of each pathogen were compared to the melt curves and associated amplicon sequences of
A.
pyogenes,
E.
coli,
S.
agalactiae,
S.
dysgalactiae,
K.
pneumoniae,
S.
uberis and
S.
aureus. The selection of a collection of bovine strains for these pathogens to be utilized as reference strains in the HRMA was deemed required due to the notable discrepancies in melt curves and sequencing between human and bovine isolates of
E.
coli and
K.
pneumoniae. Through analysis of the melt curves of 60 bacterial cultures recovered from mastitis milk samples, three interpreters verified the HRMA. The culture and sequencing results of the isolates were concealed from the three test interpreters. A 95% validation test accuracy was achieved overall because the variability in the
S.
uberis PCR amplicons made it challenging to identify the
streptococci. According to this study, differentiating between clinically significant bacterial mastitis pathogens can be accomplished quickly, effectively and affordably using broad-range real-time PCR in conjunction with HRMA.
