Current Update on Emerging Technologies and Their Clinical Relevance for Early Detection of Mastitis in Dairy Cattle
Dr. Alok Kumar Chaudhary
Assistant professor, Department of Veterinary Medicine, DUVASU, Mathura U.P. India
Abstract
Mastitis has great economic importance for Indian dairy sector; it negatively impacts several aspects of production and health of dairy animals. Apart from this, it is also a matter of public health concern. Clinical mastitis is directly affecting animal health and production, however, subclinical mastitis is major responsible for deteriorate international standards milk and milk products. For quality production of milk up to international norms, control of mastitis is the prime subject for the nation. It needs to adopt some emerging technology based diagnostic techniques for early, fast, and accurate diagnosis, which must be helpful in making an appropriate strategic plan for better treatment and control of mastitis in dairy animals.
Key words: somatic cell count, mastitis, markers, cytokines, inflammations
Introduction:
Mastitis is one of the most frequently occurring mammary gland affections in dairy animals. In this condition, mammary tissues are extremely damaged by numerous pathogenic or non-pathogenic agents through an immuno-pathological mechanism. The severity of structural damage of mammary tissues directly influences milk quantity and quality as well as animal health status. Therefore, infection of the mammary gland produced 4D effects as on mammary structure, milk, animal health state, and the environment. Mastitis is one of the most painful conditions for dairy animals, so it is now noticeable under animal welfare. Apart from these, zoonotic and antimicrobial resistance are the most prevalent issues in India due to such affected milk utilisation in the human population. Economically, it has a negative impact on several aspects of the production and health of animals. Undiagnosed subclinical mastitis milk detoriates milk quality parameters and is one of the main reason for refusal of milk from global markets. However, with clinical mastitis, it severely affects all 4D dimensions. The concept of early diagnosis is one of the only solutions to control its 4D effects, but it is the biggest challenge for the dairy industry as well as associated researchers. Recently, a holistic diagnostic approach must be made on the basis of the type of mastitis. In clinical mastitis, four affected sites must be considered for appropriate diagnosis. The first is abnormal milk (changes in color, clots, flakes of somatic cell count), the second is abnormal mammary tissues (changes in tissue color, swelling), the third is changes in animal status (body temperature, appetite, and hydration level), and the fourth is the infection level of the environment. Besides this, in subclinical mastitis, there are fewer affected sites like milk (negatively affected in milk quantity and quality parameters) and infection level of the environment. Bidirectional research is going on to find out the correct solution for early inflammatory indicators and exact identification of caustic agents’. The prime objective of this article is to compile the routine/emerging diagnostic techniques and analyse their clinical relevance for mastitis diagnosis.
- Conventional markers of mastitis
Various conventional indicative markers for diagnosis of mastitis have been established, but owing to clinical limitations and lack of specificity, some are not used in ordinary procedures. However, several fundamental tests are still using as a basis of mastitis diagnosis. Some fundamental traditional indicative markers are as follows:
(i) Indicative markers based on organoleptic proprieties of milk:
It is the traditional basis for ensuring the quality of the milk. The traditional method is based on the organoleptic proprieties of milk such as colour, flavour, taste, smell, and texture. Any change in the organoleptic properties of milk is a strong indicator of milk quality deterioration. These types of indicative markers provide consistent information for abnormal milk quality, but they cannot ensure whether the source of the milk contamination is internal or external. Apart from this, it can’t possible to differentiate the exact cause of the milk deterioration.
(ii) Indicative markers based on physo-chemical properties of milk:
Milk’s physiochemical qualities are directly linked with dairy cows’ health and production. Normal milk with average physiochemical characteristics is secreted by a healthy udder with a well-balanced feeding system. Any anatomical or functional change in the mammary gland causes aberrant milk to be produced with altered physiochemical characteristics. Density, temperature, and freezing point are the most dependable physical markers for assessing the cellular structure and functional activity of the mammary gland. Milk fat, protein, lactose, SNF, and TS are chemical-based indicators; these markers examined by Lacto scan milk analyzer and instruments. Clinical relevance of these indicators is also limited. They are also unable to identify the exact cause of altered structural and functional cellular activity of mammary gland
(iii) Indicative markers based on pro- inflammatory agents
When an antigenic agent enters in the mammary gland, its defence system is engaged, and a slew of pro-inflammatory mediators are released. These mediators are currently being looked at as possible early markers of inflammatory chain reactions. These mediators are easily traceable in serum, and some of them may now be tested in milk using an ELISA-based kit. Serum amyloid A (M-SAA), haptoglobin (HP), and lactoferin are the most frequently used indicators. Furthermore, other acute phase proteins such as catheicidins, chemo attractants, and cytokines (IL-1 TNF alpha) have been used to detect inflammatory responses. They are reliable and fast indicative markers for inflammatory reaction but it also have clinical limitations it can’t explain source of the inflammatory reaction.
(iv) Indicative markers based on enzymes:
Pro- inflammatory mediators started signaling for active phygocytic cells to reach and destroy the target invaders. These cells released several types of enzymes that help in engulfing and killing the microorganisms. These enzymes have now become reliable inidactive markers for enzymatic reaction against any invaders in the body. In recent literature, N-acetyl-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) are the two most commonly measured enzymes. The serum concentration of these enzymes directly reflects the rate of tissue destruction in the body. However, it also has clinical limitations; it can only quantify the rate of cellular damage, but it cannot give any specific ideas about the etiology of involvement. It can be measured by colorimetric and fluorometric based assays from raw milk at the farm level.
Fig: 2 Summery of conventional and molecular indicative markers for mastitis
(v) Indicative markers based on somatic cell count (SCC):
Somatic cells are basically a mixed density of epithelial and leukocyte cells and they are normally present in milk with an average ratio of 25 % and 75 % respectively (Addis et al, 2016). When the level of infection or cell distraction increased in the mammary gland, the number of epithelial cells and leukocyte cells increased, causing the average ratio of epithelial cells and leukocyte cells to change. Direct microscopic, kits, somatic cell counters, and differential somatic cell counters are the most common technique to monitoring somatic cell count. It is one of the most reliable and authenticated tools for screening of mastitis at herd level, especially for subclinical mastitis. In general, SCC values are above 200,000 cells/ml of milk. On the basis of SCC concentration, several laboratorial and field level tests have been implemented with different protocols and principles. CMT also depends on the number of somatic cells. Its principle is addition of a reagent with sodium lauryl sulfate (detergent), to disrupt somatic cell membranes and promote lysis and release of DNA, forming a gel that is visible to the naked eye. However, it only provides quantitative results and it also prone to false positives due to subjective interpretation.
(vi) Indicative markers based on electrolytes:
Excessive cellular destruction and a weak milk/blood barrier in mastitis lead to changes in actual ionic dynamics in vascular components. Vascular concentrations of sodium, potassium, calcium, magnesium, and chloride ions increase, while the concentration of potassium ions decreases due to loss of cellular potassium. These changes modify the electro-conductivity (EC) of milk and increase pH levels. These changes are used as an indicative marker to identify abnormal milk proprieties. Recently, some robotic milking machines have been assembled with electrical conductivity measurement techniques. However, it is not a reliable or sensitive parameter for conclusive diagnosis of mastitis
- Infectious mastitis indicative markers
Identification of infectious agents of mastitis is based on some distinct phenotypic and genotypic characteristics of particular agents, and these act as diagnostic markers that would be identified by different techniques which are as follows.
- Indicative markers based on phenotypic characteristics:
Phenotypic characteristics of infectious agents are one of the basic criteria for the identification of microorganism groups. Culture and isolation techniques are the basic methods for the identification of microbes. But the main clinical limit of this technique is that it is not easy to isolate and identify all the microbes, especially viruses and fungus. Another major issue is that it is only valid for live microbes; if the causative agent dies during transport/sample storage, it cannot be identified. Apart from this, it has capacity only to detecting viable cells. Apart from this, it also requires a specially trained person and is time consuming. Recently, some other methods have been used to determine bacterial and bacterial strains. MALDI-TOF MS (matrix-assisted laser desorption ionization–time of flight) mass spectroscopy based techniques can be performed to determine bacterial and bacterial strains within a few minutes (Matuozzo et al., 2020). Never the less, the ability of MALDI-TOF MS in identification is limited to specific spectra databases of the existing bacterial protein profiles, and this technology is still too costly to be widespread in diagnostic laboratories.
(ii) Indicative markers based on antigenic/antibody characteristics:
Various antigen /antibody specific ELISA kits for microbial identification have been developed, like S. aureus (Fox and Adams, 2000), Listeria monocytogenes (Kalorey et al., 2007). This technique is based on antigen antibody reaction by using ELISA based kits. These ELISA based kits are often very commonly used because of their speed, simplicity, relatively low cost, and availability. Its clinical application is that it cannot cover all types of microbes in single test and most of the etiological agents until not develops this type of kit.
(ii) Indicative markers based on genetic characteristics:
Recent developments in genomic studies helps in the development of various genetic material based diagnostic techniques. This technique is highly sensitive and reliable for identification of the etiological agents of mastitis. This type of test can easily detect growth-inhibited, viable or non-viable bacteria (Klass and Zadokes, 2018). Genetic material of common mastitis inducer agents is given in Table-1 and is used in various techniques for identification of bacteria.
Table-1 Genetic markers of infectious mastitis inducers
Various technologies have been developed and certified to diagnose single bacteria or groups of bacteria in milk sample. Some important techniques are discussed as:
(a) PCR techniques:
PCR one of the gold standard tests for the diagnosis of etiologically based mastitis. With these techniques, the presence of bacterial DNA in mastitic milk can be easily diagnosed at strain level. As compared to the culture method, it is less time consuming and has been extensively used for more than a decade. The clinical limitation of PCR is that it based on the detection of particular bacterial DNA. However, mastitis is known for the involvement of multiple etiological agents. A single PCR always has a chance to fail to detect real damaging agents. Another limitation is that PCR does not work on live of dead cells and is unable to quantify DNA molecules. It indicates PCR is unable to quantify the infection level but it more accurately detects any target microorganisms in a given sample.
(b) Multiplex PCR:
Due to multiple etiological involvements in mastitis, a new multiplex PCR technique helps to solve a big issue. In this technique, many primer pairs are mixed together in the same PCR reaction for screening of samples for the presence of different pathogens, or to search for the presence of different virulence genes within the same isolate. Identification of the mastitis pathogens becomes easy, rapid, and standardized as compared to conventional PCR but it cannot quantify the infection level at the time of sampling. It also has a drawback, which is competition between different sets of primers for PCR substances like dNTPs and Taq polymerase, which reduces the sensitivity.
(c) Real-time PCR:
It’s used to offer additional benefits compared to bacterial culture and conventional PCR. It is not only faster (within 4 h after) and more sensitive (100%) but also safer for the workers and the environment (no ethidium bromide is used), post reaction handling is not needed (no agarose electrophoresis), and better visualization and digitization of the results, which enables documentation and data exchange with other teams. In addition, RT-PCR can be applied for the quantization of pathogens in infected milk by measuring the intensity of the produced fluorescence from the reaction. It is more costly, so it cannot be afforded at an individual level.
(d) -Microarray assay:
Microarray technology is based on the hybridization of hundreds of target genes loaded on the microarray chips followed by their visualization through being exposed to sequence complimentary DNA probe conjugated with fluorescence or chemiluminescent stains. .The arrays are slides on which robotically added DNA is spotted to permit genome analysis of the investigated pathogens. The use of microarray assay is a technically demanding and costly technology which is usually applied in research laboratories.
(e) –Loop-mediated isothermal amplification (LAMP):
This method is faster than PCR, less expensive, highly specific for the target sequence and less demanding in terms of the quality of the template and complex instrumentation. Ultimately, as an isothermal amplification technique, it could be implemented on field settings, requiring only a water bath or heat block for the reaction. LAMP assays have been described for mastitis pathogens such as S. aureus, S. agalactiae, and S. uberis from bovine mastitis milk samples
(f) – Next-generation sequencing (NGS):
It is a new genotyping method to identify mastitis infectious agents, as NGS is becoming more available and affordable. This method allowed the successful detection of multiple bovine pathogens in clinical samples, including some additional pathogens missed by routine techniques because the specific tests needed for the particular organisms were not performed .This result demonstrates the feasibility of the approach and indicates that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory and that, likely in the near future, NGS sequencing can be used as a tool in the routine identification of mastitis related microorganisms.
(C) Indicative markers based on emerging technologies
Recent advances in omic and nanotechnologies have opened the development of a new class of diagnostic markers and artificial intelligence based analytical tools for the diagnosis of animal health states, udder health, and milk quality and quantity on single button operating systems. In this series, biosensors, biochips, and microRNA/miRNA are some of the fastest developing tools worldwide.
- Biosensors for detecting bovine mastitis:
A biosensor is a device with micro-systems that combines a biological element called bioreceptors with a physical transducer and sensor. There are several types of transducers that have been used, but for pathogen detection, electrochemical, ,optical , fiber optic surface plasmon resonance (SPR) ,piezoelectric , gravimetrical, magnetic, and acoustic sensors are extensively used .Biosensors are biological sensors which use bio-receptors like antibodies, nucleic acid, enzymes etc. to produce a signal after combination with transducers. Nowadays, some of the biosensors have already been developed for detecting bovine mastitis, for example. For example, an electro biochemical sensor developed by Pemberton et al., 2001 and a competitive biosensor assay using surface plasmon resonance were developed to discriminate between sub-clinical mastitic and non-mastitic milk Akerstedt et al., 2006.
- Biochips for detecting mastitis:
“Biochip” are so called as ‘laboratory-on-a-chip’ or microfludics, having the capacity to be used as a diagnostic and are already used for the detection of bovine mastitis viz. Lee et al., 2008 developed a biochip that integrated DNA amplification of genes that are specific to seven known mastitis-causing pathogens; a micro fluidic device that incorporated solid-phase extraction and NASBA has been reported for the identification of low numbers of E. coli which can detect the most common species of mastitis-inducing pathogens, namely; Corynebacterium bovis, Mycoplasma bovis, Staphylococcus aureus, and the Streptococcus spp. Strep. agalactiae, Strep. bovis, Strep. dysgalactiae, and Strep. uberis, within a few hours Dimov et al., 2004.
- Circulating miRNA:
MicroRNA/miRNA are 22 nucleotide long non coding RNA act as transcriptional and post-transcriptional regulation during expression of genes They are complimentary to the 3’UTR region of mRNA and thus participate in gene regulation .Bioinformatics analysis revealed that 89 putative miRNA target sites present in 18 mastitis candidate genes. It was also experimentally proved already that Bta-mir-142* have target site on SAA3 mastitis candidate gene expressed in bovine mammary tissues Gue et al., 2007.
(d) Other useful technologies:
For example, advancements in proteomics tools, such as two-dimensional gel electrophoresis (2D-GE) and mass spectroscopy (MS) (Smolenski et al., 2007) helped to identify various proteins expressed during mastitis. These methods can be applied to detect the marker proteins from cases of mastitis, particularly acute, subacute, and chronic mastitis. Changes in the milk proteome during bovine mastitis could provide a significant insight into variation in protein composition that could lead to the discovery of protein biomarkers for bovine mastitis. Furthermore, by applying proteomic analyses, it may be possible to identify specific and potential protein targets for therapies and immune manipulations against bovine mastitis .The thermal camera used in this method absorbs infrared radiation and based on the amount of heat generated, the images are produced and generated. The ability of IRT to detect increased body temperature has made it possible to assess many aspects in animal industry. As measured by the IRT after endotoxin infusion into the mammary gland caused an increase of 2.3ºC of temperature as measured by the IRT (Scott et al., 2000).
Conclusion:
Mastitis is one of the biggest challenges for the dairy sector in India and one of the biggest hurdles to doubling the former income. Early and exact diagnosis is one of the single solutions to control this type of economical digester. Policymakers should give emphasis to establishing a recent diagnostic technology based regional mastitis surveillance centre with all over India. Associated workers must assume greater responsibility for educating farmers about the economic and public health consequences of their actions.
Acknowledgements:
- Umesh Dimri, Principle Scientist Division of Medicine, IVRI Bareilly
- Mukesh Kumar Srivastava, Assistant Professor, Veterinary Medicine, DUVASU, Mathura U.P
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https://www.frontiersin.org/articles/10.3389/fbioe.2019.00186/full
