Key genes involved in wax biosynthesis, transport and deposition identified from the model system Arabidopsis.
\r\n\tThe main idea for any data or information processing system for those aspects of aggregating the data or computing the hierarchy of various process elements is that they should not only be machine-readable but also machine-understandable. Moreover, an adequate knowledge-based system is perceived to be, on the one hand, understandable by people, and on the other hand understandable by the machines.
\r\n\tAs devices become smarter and produce data about themselves, it will become increasingly important for scientists to take advantage of more powerful tools and/or data integration techniques to help provide a common standard for information dissemination across the different platforms. To this end, the content of this book shows that technologies such as the semantic web, machine learning, deep learning, natural language processing, and learning analytics which encompasses the wider spectrum of the Linked Open Data (LOD) are of paramount. Therefore, the work presents two main drivers for the Linked Open Data technologies: (i) encoding knowledge about specific data and process domains, and (ii) advanced reasoning and analysis of the big data at a more conceptual level.
\r\n\tThis book intends to provide the reader with a comprehensive overview of the current state-of-the-art within the Linked Open Data and the benefits of the methods – ranging from the semantics-aware techniques that exploit knowledge kept in (big) data to improve data reasoning (big analysis) beyond the possibilities offered by most traditional data mining techniques.
In the current era of increasing uncertainties in crop production, emerging constraints and risks demand technical and technological advances in the agricultural sector, and integrative approaches, such as Climate Smart Agriculture (CSA), to address the interlinked challenges of food security and climate change. While maintaining food security is a major challenge for future, the possible solution is to enhance crop productivity along with nutritional security. However, this stance is remarkably limited by the different abiotic as well as biotic environments, where the crops grow and develop.
Drought, excess water (flooding), extremes of temperatures (cold, chilling, frost, and heat), salinity, high and/or low light, mineral deficiency, and toxicity are the common abiotic stresses for crop production. These stresses alter plant metabolism, growth, development, and in extreme cases cause the cessation of vegetative and reproductive growth. Some of the abiotic stresses such as drought, high temperature and salinity can influence the occurrence and spread of biotic agents like pathogens, insects, and weeds . In crops like tomato, cucurbits and rice, temperature is one of the most important deciding factors for the occurrence of bacterial diseases . Temperature can also alter the incidence of vector-borne diseases by modifying spread of vectors.
But, in their natural environment, plants face combination of stresses, especially under the changing climate scenario. The effect of stresses would be more pronounced under combined (biotic and abiotic) stresses , while simultaneous occurrence of abiotic and biotic stresses are more destructive to crop production . Hence, there exists a need now, to look for common traits that can contribute for plant adaptation to such multifarious stressful conditions and sustain crop productivity as well. In this scenario, it is desirable to have a single trait that can confer tolerance to multiple (abiotic and biotic) stresses. Cuticular waxes, a major component of plant cuticle covering all the aerial parts of the plants, can be considered as an important trait for combined stress resistance.
The cuticle is a unique structure developed by land plants during the course of their evolution from an aquatic to a terrestrial lifestyle . The primary role of this lipophilic layer, comprising cutin and cuticular waxes, was to limit non-stomatal water loss by functioning as a physical barrier between the plant surface and its external environment . Development of a cuticular barrier is one of the major adaptive mechanisms for survival and growth of plants under water limiting terrestrial conditions . As the primary barrier between the aerial surface of plants and the external environment, the cuticle also protect the plants from mechanical rupture or injury, toxic gases and ultra violet radiation [8, 9, 10]. The cuticle also has notable roles associated with growth and developmental processes like preventing epidermal fusion by establishing normal organ boundaries , and phytohormone homeostasis . The cuticle and its components are known to play essential roles as signaling molecules for pathogens and for the plants themselves . Another important role is in fruits, where it influences quality, defense and post-harvest shelf life . In fruits, water retention  firmness  and its responses to physical and biotic stresses are also influenced by the cuticle .
The cuticle is composed of a covalently linked scaffold of cutin and a mixture of soluble cuticular lipids (SCL), called as waxes [10, 18]. Structurally, cutin is made of covalently cross linked C16 or C18 oxygenated fatty acids and glycerol, forming the most abundant structural component of the cuticle . The waxes within the cuticle function as an actual barrier against the diffusion of water or solutes [20, 21]. The waxes occur in two layers; forming two distinct physical layers called intra- and epi-cuticular waxes . The former is dispersed within the cutin polymer while the epi-cuticular wax is deposited on the outer surface as crystals or films [22, 23]. This outermost layer can be physically stripped off the surfaces using aqueous glue [23, 24]. These waxes are composed of a variety of organic solvent-soluble lipids; consisting of very-long-chain fatty acids (VLCFA) and their derivatives. The major composition of VLFCAs are alkanes, wax esters, branched alkanes, primary alcohols, alkenes, secondary alcohols, aldehydes ketones, and unsaturated fatty alcohols, as well as cyclic compounds including terpenoids and metabolites such as sterols and flavonoids [19, 25, 26, 27]. Wax composition varies with crop species and differs in their functions and responses to biotic and abiotic environments .
As per recent studies, intra-cuticular waxes form the primary transpirational barrier and the contribution of epi-cuticular waxes as a transpirational barrier depends on the species-specific cuticle composition . In species like Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera deliciosa, and Schefflera elegantissima, intra-cuticular wax pre-dominantly act as a transpirational barrier while in Citrus aurantium, Euonymus japonica, Clusia flava, and Garcinia spicata, both intra- as well as epi-cuticular waxes had equal contribution as transpirational barriers . A study from Prunus suggests that intra-cuticular waxes of the cuticle form the actual transpirational barrier  and not epi-cuticular waxes .
The cuticular waxes confer diverse surface properties to plant parts, which actually play the key role in controlling non-stomatal water loss and gas exchange, and protection from external environment. Leaf cuticular wax amount and crystal morphology regulated post-harvest water loss from leaves . Epi-cuticular wax films give glossy appearance to leaves and fruits, while wax crystals (β-diketones) conferred dull, glaucous appearance to leaves and stems . The thickness  composition and properties of the waxes vary with crop species and are found to be induced under diverse stressful conditions . These differences reflect their functions and responses to biotic and abiotic environments . Importance of cuticular wax accumulation in plant resistance to both biotic as well as abiotic stress conditions is now well documented [12, 33, 34].
One of the most important roles of the waxes is to protect the plant surfaces from excessive solar and ultraviolet (UV) radiations. Cuticular waxes scatter UV-B radiation  and was demonstrated in apple . As per studies from model systems as well as crops, increased cuticular wax biosynthesis improves drought stress resistance . In rice, wheat, barley and sorghum, grain yield under water limiting conditions have positive correlation with wax content [38, 39, 40, 41] . Hence, in crop plants, higher cuticular wax content is a promising trait for stress resistance as well as yield under water limiting conditions . In mulberry, increasing wax load is useful to manage post-harvest water losses . In barley, cuticular wax components act as a barrier to water loss and contribute to salt stress resistance . Heat stress resistance is also positively correlated with wax accumulation in bahia grass . Under heat stress, the wax load in sorghum was correlated with its ability to maintain the canopy temperature cool, resulting in reduced water loss . Similarly, pea varieties with thicker wax load also exhibited lower canopy temperature, thereby limiting water loss and associated heat stress .
Cuticular waxes play an important role in preventing non-stomatal water loss during drought and high temperature stress, as well as enabling frost avoidance. Such climatic stressors can induce a heavier wax load and change the chemical composition of waxes by accumulating longer aliphatic compounds on plant tissues . Drought increases stiffness and quality of the plant cuticle under climate change . Similarly, the leaf cuticular surface is the first barrier blocking destructive ice penetration into the leaf cells in freezing avoidance mechanisms . Using a hydrophobic film, Wisniewski et al.  showed the importance of the epi-cuticular hydrophobicity enabling avoidance of freezing in sensitive plants. The critical nature of the cuticular layer in frost avoidance of corn is also clearly demonstrated . Freezing avoidance is the only mechanism of frost resistance in sensitive plants. In fact, the first demonstration of a transgenic organism in agriculture was the alteration of the cell wall protein secondary structure on ice nucleating bacteria, Pseudomonas syringae and Erwinia herbicola, which then prevented ice nucleation across the cuticle and avoided leaf damage [52, 53]. In future, injury due to frost stress will be more, not less under global warming . Hence, a better understanding of stress-induced wax modification among crop plants holds promise to cope with climate change.
The cuticle and its components act as signaling molecules to favor fungal growth and development, and infections in plants [55, 56]. Surface waxes act as cues to activate fungal developmental processes like appressorium formation, pre-penetration processes, etc., in crop plants like avocado, wheat, rice, maize and peanut [13, 57, 58, 59]. However, the hydrophobic nature of the cuticle also renders it a barrier for bacterial as well as fungal pathogens , a desirable trait for disease resistance. Waxes are known to protect lotus from pathogen infection . It repulses pathogen spores and atmospheric pollutants like acid rain and ozone . Another role of waxes is in plant-insect interaction; to attract or to serve as a deterrent . It prevents insect attachment to plant surface oviposition and feeding [63, 64] and hence confer tolerance to insects in crop plants [65, 66].
Studies in Arabidopsis and subsequently, barley, rice and tomato systems have significantly contributed for the elucidation of the complex regulatory pathways underlying the biosynthesis, transport and deposition of wax components on plant surfaces [26, 27, 67]. Cuticular wax biosynthesis predominantly occurs in epidermal cells. The biosynthetic pathway initiates exclusively in the outer membranes of the plastids of epidermal cells where C16 and C18 fatty acids are synthesized, exported to the cytosol as acyl-CoAs and then elongated up to C34 at the endoplasmic reticulum (ER); through a series of enzymatic reactions [19, 26]. The synthesized components are subsequently transported through the apoplastic pathway and deposited on the cuticle. The key steps involved  are summarized here.
The de novo fatty acid biosynthesis initiates with the synthesis of malonyl-CoA. It is initiated with the transfer of a bicarbonate derived CO2 molecule to the biotin moiety of a biotin carboxylate carrier protein (BCCP), that form N-1,2 carboxybiotin biotin carboxylate carrier protein-BCCP. The reaction is catalyzed by biotin carboxylase (BC). The CO2 is further transferred to acetyl-CoA by carboxyltransferases (CT). Acetyl-CoA carboxylase (ACCase), a multifunctional enzyme system then catalyzes the formation of malonyl-CoA, from acetyl-CoA , which will be subsequently used for de novo fatty acid biosynthesis.
De novo synthesis of acyl chain in the stroma of plastids is catalyzed by a series of enzymatic steps, which collectively forms fatty acid synthase complex (FAS). The series of reactions with the catalyzing enzymes are:
Condensation of malonyl-acyl carrier protein (manolyl-ACP) with acetyl-CoA to form 3-ketoacyl-ACP catalyzed by β-ketoacyl-ACP synthase (KAS III).
Reduction of 3-β-ketoacyl-ACP to 3-hydroxyacyl-ACP, catalyzed by 3-βketoacyl-ACP reductase.
Dehydration of 3-hydroxyacyl-ACP to trans-∆2-enoyl-ACP, catalyzed by β-hydroxy acyl ACP dehydratase.
Reduction of trans-∆2-enoyl ACP to Acyl-ACP by Enoyl ACP reductase.
This complex also includes an acyl carrier protein (ACP), a cofactor component of FAS to which the growing acyl chain remains esterified. These sequential reactions result in a fully reduced acyl chain, extended by two carbons in each cycle  through the sequential round of condensation, reduction, dehydration and second-reduction steps . Repetition of the cycle for six times generates palmitoyl-ACP (16:0-ACP), where the condensation reactions are catalyzed by KAS I. One final cycle reaction between palmitoyl-ACP and malonyl-ACP utilizes KAS II to generate stearoyl-ACP (18:0-ACP). These products are further processed by stearoyl-ACP desaturase (introduce double bonds), plastidial acyltransferases, and acyl-ACP thioesterases (hydrolases). The fatty acyl-ACP thioesterases (FATA and FATB) hydrolyzes the C16-C18 acyl-acyl carrier proteins to generate fatty acids, which are then exported out of the plastids to undergo modifications in the ER .
The C16 and C18 compounds, hydrolyzed by acyl-ACP thiosterases are activated into C16- and C18-CoA by long chain acyl-CoA synthetases (LACSs) and exported to the ER. The C16 and C18 acyl-CoA then act as a substrate for fatty acid elongase (FAE) complex, localized on the ER, which adds two carbons successively to form VLCFAs with C26-C34 chains. FAE complex are heterotetramers of independently transcribed, monofunctional proteins. They operate a reiterative cycle of four reactions catalyzed by
β-Ketoacyl-CoA synthase (KCS) that catalyze the two carbon condensation to acyl-CoA.
β-Ketoacyl-CoA reductase (KCR) that catalyze the reduction of β-ketoacyl-CoA.
β-Hydroxyacyl-CoA dehydratase (HCD) that catalyze the dehydration of β-hydroxyacyl-CoA.
The elongated products are further modified to produce wax components i.e., to primary alcohols, alkyl esters, aldehydes, alkanes, secondary alcohols, ketones and free fatty acids, via two pathways (i) acyl reduction pathway (generates primary alcohols and wax esters) and (ii) decarbonylation pathway (generates alkanes, aldehydes, secondary alcohols, and ketones).
Acyl-reduction pathway: fatty acyl-CoAs are converted into primary alcohols catalyzed by fatty acyl-CoA reductase (FAR) through an intermediate aldehyde . A bi-functional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzyme, WSD1 condenses the generated fatty alcohols and C16:0 acyl-CoA into wax esters .
Decarbonylation pathway: acyl-CoAs are reduced to aldehyde intermediate by FAR, which are subsequently decarbonylated into alkanes, catalyzed by aldehyde decarbonylase. Stereospecific hydroxylation of alkanes catalyzed by midchain alkane hydroxylase 1 (MDH1) give rise to secondary alcohols, and oxidation of these alcohols form corresponding ketone . Additional hydroxylation and oxidation reactions lead to the esterification of secondary alcohols with fatty acids and formation of diols, hydroxyl ketones and diketones .
The wax components generated are then transferred from the ER to the plasma membrane (PM) through Golgi and trans-golgi network mediated vesicle trafficking or non-vesicular trafficking . Further, adenosine triphosphate binding cassette (ABC) transporters in the plasma membrane (homodimers and heterodimers) export the wax components to the epidermal surface . Lipid transfer proteins (LTPs) like glycosylphosphatidylinositol (GPI)-anchored LTPs (LTPGs), attached to the outer surface of the plasma membrane are also directly or indirectly involved in wax export . A brief representation of wax biosynthesis, transport and deposition with key genes, is presented in Figure 1 (adapted from [19, 26, 27, 32, 69, 71]).
Early studies in barley mutants with little or no wax on aerial plant parts, called glossy or glaucous were termed as eceriferum (cer), where cera means wax and ferre means to bear . Subsequently, the wax defective mutants in Arabidopsis with bright, shiny, or glossy stems or leaves were also termed as eceriferum (cer) . The wax locus from maize and Brassica napus is termed as glossy . With the help of forward genetic screens using wax defective mutants and reverse genetic approaches [77, 78], considerable progress has been achieved in understanding wax biosynthesis, transport and deposition. Table 1 gives an overlook of the key genes involved in wax biosynthesis, transport and deposition identified from the model system Arabidopsis.
|Cuticular wax biosynthesis|
|ACC1||Acetyl CoA carboxylase||Synthesis of malonyl CoA substrates|||
|FATB||Acyl acyl carrier protein thioesterase||Supply of saturated fatty acids for wax biosynthesis|||
|CUT1 /CER6/KCS6||VLCFA condensing enzyme (β-ketoacyl-CoA synthase)||Regulation of VLCFA biosynthesis/elongation of 24C fatty acids|||
|CER1/CER22||Aldehyde decarbonylase||VLC alkane biosynthesis|||
|KCS1||β-ketoacyl-CoA synthase||Elongation of 24C fatty acids|||
|KCS20; KCS2/DAISY||3-ketoacyl-coenzyme A synthase||Required for VLCFA elongation to C22|||
|LACS1/CER8; LCAS2||Long chain acyl CoA synthetase||Synthetase activity for VLCFAs C20-C30|||
|KCS9||3-ketoacyl-coenzyme A synthase||Elongation of C22-C24 fatty acids|||
|WAX2/YRE/FLP1/CER3||Aldehyde‐generating acyl‐CoA enzyme||Required for synthesis of aldehydes, alkanes, secondary alcohols, and ketones; biosynthesis of cuticular membrane||[76, 87]|
|CER10||Enoyl-CoA reductase||Biosynthesis of VLCFA|||
|CER4/FAR3||Alcohol forming fatty acyl CoA reductase||Formation of C24:0 and C26:0 primary alcohols|||
|CYP96A15 (cytochrome P450 enzyme)||Midchain alkane hydrolase||Formation of secondary alcohols and ketones (stem cuticular wax)|||
|WSD1||Wax ester synthase/diacylglycerol acyltransferase||Wax ester biosynthesis|||
|PASTICCINO2 (PAS2)||3-hydroxy-acyl-CoA dehydratase||VLCFA synthesis in association with CER10, an enoyl-CoA reductase|||
|KCR1||β-Ketoacyl-CoA reductase||Required for VLCFA elongation|||
|CER2||BAHD acyltransferase||Fatty acid elongation beyond C28|||
|CER17 (ECERIFERUM1)||Acyl-CoA desaturase like 4||n-6 desaturation of very long chain acyl-CoAs|||
|Transport and deposition|
|AtWBC12/CER5||ATP binding cassette (ABC) transporter||Transport of cuticular waxes|||
|LTPG1||Lipid transport protein||Cuticular wax export or accumulation|||
|ABCG11/WBC11/DESPERADO||ATP binding cassette (ABC) transporter||Secretion of surface waxes in interaction with CER5||[73, 95]|
|LTPG2||Lipid transport protein||Cuticular wax export or accumulation|||
|GLN1, ECH||Vesicle trafficking|||
While the complex wax biosynthesis and transport pathways are well determined, the information on underlying regulatory mechanisms is still fragmentary. There is limited information that these processes and their candidate pathway genes are influenced by developmental factors. The cuticle development is an intrinsic part of cell developmental processes like organ development, cell partitioning, etc. . PAS2, acy-CoA dehydratase, regulating the synthesis of VLCFA during wax biosynthesis in the epidermis is essential for proper cell proliferation during development . Wax deposition is also known to occur in an organ-specific manner during its development and is influenced by diverse environmental conditions as well . The available information on the exact developmental regulation of wax biosynthesis is however, limited. As per evidences from leek (Allium porrum L.), wax accumulation and elongation activities are highly induced within a defined and an identifiable region of leaf . The expression of plastidial fatty acid synthase (FAS), FAEs that regulate elongation of long-chain fatty acids in the microsomal membranes and acyl ACP-thioesterases are probable targets of developmental regulation, depending upon the need to produce fatty acid precursor pools . Some of the key genes involved in wax biosynthesis are also affected by defects in the organization of organelles, especially the ER. A mutation of PEX10 (peroxisome biogenesis factor 10) in Arabidopsis, which disrupted the ER network, in turn lead to mislocalization of CER4, CER1, SHN1 and WAX2, affecting cuticular wax biosynthesis .
There is increasing evidence to show that wax biosynthesis and its pathway genes are regulated at transcriptional, post-transcriptional and translational levels [26, 100]. A wide range of abiotic factors like light, water, temperature, salinity etc., influence wax biosynthesis and deposition. An increase in cuticular wax content is observed in bean, barley and cucumber on exposure to UV-B light . In cotton, enhanced UV-B radiation specifically increased the epicuticular wax load on the adaxial surface of leaves . There is an also an up-regulation of wax biosynthetic genes in salt tolerant rice genotypes under stress . Although the underlying mechanisms have not been well explored in the above conditions, there is sufficient information on the influence of drought or moisture stress on wax biosynthesis in plants. A significant increase in wax load in Arabidopsis plants subjected to water stress is indicative of its regulation under drought . In crops like rice, wheat, tobacco, alfalfa, peanut and cotton, etc., an increase in cuticular wax accumulation was observed under moisture stress condition . Drought induced accumulation of wax biosynthesis is positively correlated with drought tolerance in crops like oat, rice, wheat and forage crops, etc. [104, 105, 106, 107].
The transcript levels of several genes involved in wax biosynthetic pathways are regulated in response to abiotic stresses. FAR5, a fatty acyl CoA reductase, in wheat responsible for accumulation of long chain primary alcohols of C26:0, C28:0 and C30:0 are regulated by drought, ABA and cold . The transcripts of KCS2/DAISY, a 3-ketoacyl-coenzyme A synthase required for the elongation of VLCFA are up regulated under water deficit conditions . Osmotic stress induces the expression of CER1, that regulates alkane biosynthesis; while the over expression of CER1 increased susceptibility to bacterial and fungal pathogens . Hypoxia is also known to affect total wax loads on Arabidopsis. The expression of KCS, KCR1, ECR/CER10 and PAS2, components of fatty acid elongase complex in Arabidopsis stem and leaves is affected which in turn affects the production of VLCFA precursors of wax biosynthesis. The wax synthesis genes like MAH1, CER3, CER4, WSD1, etc., and several genes associated with wax and lipid transport are also affected by hypoxia . There is also indication on the regulation of wax biosynthesis in response to cold. Acteyl-CoA carboxylase plays the essential role for cold acclimation in Arabidopsis. In sensitive to freezing3 (sfr3) mutants, with a missense mutation in ACC1, the long chain components of leaf cuticular wax were reduced and there was inhibition on the wax deposition on inflorescence stem, which rendered the plants sensitive to cold stress . Wax biosynthesis is also reported to be regulated in response to carbon dioxide (CO2) concentration. This is mediated by HIC (High Carbon Dioxide), a gene encoding a 3-keto acyl coenzyme A synthase (KCS)-an enzyme involved in the synthesis of very-long-chain fatty acids that influences stomatal development in Arabidopsis .
With the identification of several transcription factors (TFs), transcriptional regulatory mechanisms are considered to be a major contributor for the wax biosynthesis . WIN1/SHN1 (WAX INDUCER 1/SHINE1) is a TF from AP2/EREBP family initially reported to regulate cuticular wax and then cutin biosynthesis by regulating the expression of CER1, KCS1, CER2, LACS2, GPAT4, CYP86A4, CYP86A7 and HTH-like genes . SHN1 overexpression increased drought tolerance in Arabidopsis . Wax synthesis regulatory gene 1 (WR1) from rice  and SHN1 from wheat , both homologs of WIN1/SHN1 from Arabidopsis also reduced water loss and improved drought tolerance. Transcriptional repression by diurnally controlled DEWAX2 is another important for regulator of wax biosynthesis in Arabidopsis. Compared to wild type, the total wax loads in dewax2, were increased by 12 and 16% respectively in rosette and cauline leaves [118, 119]. Another candidate from AP2/ERF TF family, WRINKLED4 (WRI4) positively regulates wax biosynthesis in stems. wri4 mutants expressed 28% reduction of total wax loads in stems, although siliques and leaves were unaffected. Hence WRI4 act as a transcriptional activator to regulate the expression of LACS1, KCR1, PAS2, ECR and WSD1, to maintain the levels of 29C long alkanes, ketones and secondary alcohols in stems . MYB94, regulate the expression of wax biosynthetic genes like WSD1, KCS2/DAISY, CER2, FAR3 and ECR to activate cuticular wax biosynthesis and is up regulated by drought and ABA. This also conferred tolerance to drought stress in Arabidopsis and Camelina . MYB96, an ABA responsive TF also regulates wax biosynthesis under drought . In Camelina, MYB96 activated the expression of wax biosynthetic genes KCS2, KCS6, KCR1-1, KCR1-2, ECR, and MAH1 which resulted in high levels of alkanes and primary alcohols and improved drought tolerance . MYB96 acts as a component of plant disease resistance, through salicylic acid mediated signaling . Both MYB94 and MYB96 share a common region containing MYB consensus motifs in the promoter of their target wax biosynthetic genes . Hence MYB94 and MYB96 have an additive role on plant cuticular wax biosynthesis and under drought and ABA conditions.
In addition, to transcriptional regulation, wax biosynthesis is regulated by other events. Expression of CER3/WAX2/YRE, an aldehyde-generating acyl-CoA enzyme in the wax biosynthetic pathway is regulated by CER7, a core RNA processing and degrading exosomal subunit. CER7 regulates WAX2 transcript levels by degrading a specific mRNA species encoding its negative regulator . Many of such regulators have been identified from model systems as well as crop species and a brief overview of the key regulatory events and their targets has been presented in Figure 2.
Under field conditions, crops encounter multiple biotic and/or abiotic stresses simultaneously at different stages of developments. Cuticular waxes have a direct role in multiple stress tolerance in crops . In cucumber, wax biosynthesis has been shown to have key roles in influencing the plant responses to biotic as well as abiotic stresses . In sorghum, genes regulating leaf waxes have critical role in regulating tolerance to drought and heat stress . Considering the relevance of cuticular waxes under diverse biotic as well as abiotic stressful conditions, as discussed above and under combined stress conditions, it can be an ideal trait to tackle multiple stresses in crop plants.
Being the outermost layer of plant cuticle, the epi-cuticular wax can serve as a first line of physical defense against pathogens and herbivores. However, increasing thickness and hydrophobicity of the cuticle through over-deposition of the wax may not necessarily increase the resistance of the plant against biotic stresses. The composition and structure of wax in the cuticle can constitute the source of signals for the foreign invaders and for the plants themselves. Thus, the roles of cuticular wax could be multifunctional and can vary not only for various plant species but also for different kinds of pathogens. Functional study of the DEWAX gene, a negative regulator of wax biosynthesis in Arabidopsis, is a good example of this complexity. The dewax mutant line in Arabidopsis, with increased epicuticular wax and decreased cuticular permeability, showed susceptibility to the fungal pathogen Botrytis cinerea, but resistance to the bacterial pathogen Pseudomonas syringae . Moreover, DEWAX overexpressing lines in Arabidopsis and Camelina showed inverse defense modulations to B. cinerea and P. syringae as compared to dewax mutant in Arabidopsis .
Wax and cutin components in the plant cuticle could function in pattern- and effector-triggered immunity (PTI and ETI) and could serve to generate local and systemic acquired resistance against numerous pathogens . During plant-pathogen interaction, the plant cuticle can be affected by enzymes synthesized and secreted by the pathogens. Many fungal pathogens synthesize and secrete hydrolytic enzymes (for example, cutinases, esterases and lipases) at the early stage of infection that directly target the cuticle [128, 129, 130, 131]. Fusarium oxysporum secretes cutinases that degrade cutin layers in the cuticle and generates cutin monomers that support fungal adherence to the host plant and facilitate the initiation of infection . Hexadecanediol, a cutin component in rice can facilitate spore germination and differentiation for pathogenic fungi Magnaporthe grisea and B. cinerea . Presence of a very-long-chain C26 aldehyde (a wax component) was important for the barley powdery mildew fungus (Blumeria graminis) to initiate infection in host plant species. Germination and appressorial differentiation of B. graminis were strongly prohibited in aldehyde free glossy11 mutant in corn. Spraying of n-hexacosanal (C26-aldehyde) or wax preparation from wild-type corn can restore the conidial formation and differentiation .
Plant can also recognize the attachment of pathogens and activate defense responses against them, in which pathogen-infection generated plant products, such as cutin monomers or cell wall oligosaccharides, can act as signaling molecules . Defense responses in plants are often manifested as alternations of the cuticle. Colletotrichum acutatum infection in citrus resulted in increased lipid synthesis in the epidermal cell and increased deposition of those lipids in cuticle, the process eventually changes the structure of the cuticle . Cuticular biosynthesis was also found to be up-regulated in tomato fruit following infection by fungal pathogen C. gloeosporioides .
Cuticular permeability plays a vital role in almost all plant-pathogen interactions. A more permeable cuticle can lead to either resistance or susceptibility to pathogens. Elevated deposition of cuticular wax as well as the presence of hydrophobic wax components (e.g., very-long-chain alkanes or ketones) can make a cuticle less permeable. Mutation or overexpression of genes that diminish biosynthesis of various wax components can generate the opposite effect. There are number of wax-deficient mutant and transgenic lines in Arabidopsis and other plant species with diminished cuticular permeability showed resistance to the fungal pathogen B. cinerea [34, 127]. However, the phenomenon is not true for all wax deficient plant lines. Wax and cutin deficient acp4 and gl1 mutants in Arabidopsis displayed increased sensitivity to B. cinerea [135, 136]. Mutations in SHINE transcription factors in other studies also showed alteration in cuticular wax accumulation, and susceptibility to B. cinerea infection [137, 138].
Epicuticular wax also plays important roles in plant interaction with insects and herbivores. Flowering plants have evolved with cuticular wax of various forms, sizes and structures that are either enabling the attachment and movement of pollinating insects, or reducing the attachment of herbivorous insects and pests on the plant surfaces. Reducing the attachments of herbivores on plant surfaces is a part of a plant defense strategy against herbivores.
Most plant body surfaces are covered with a two-dimensional (2D) epicuticular wax film of various thicknesses. In many species, wax film is protrudes with three-dimensional (3D) wax crystals. Wax crystals can generate various shapes as revealed by electron microscopic analysis, such as rodlets, threads, platelets and tubules . The complexity of these various shapes originates from the molecular self-assembly of various wax components, in which morphology of those crystals is also correlated with the presence of specific chemical components in the wax [139, 140]. Many experimental studies and reports from various plants species (for example, from genera Eucalyptus, Pisum, Brassica) have shown that 3D wax crystals have protective functions against insects, in general, including the herbivorous insects . Studies with Eucalyptus species in canopy found that glaucous juvenile leaves containing high quantities of wax crystals were less prone to herbivorous infestation as compared to the glossy adult leaves . Feeding rates of flea beetles, Phyllotreta cruciferae, on low-wax glossy (eceriferum, cer) Brassica napus mutant lines were much higher as compared to the wild-type B. napus . Cuticular surfaces with wax crystals also interferes with the attachment, locomotion and foraging behavior of predatory insects and parasitoids [65, 144]. Pisum sativum lines with higher prevalence of crystalline epicuticular wax (CEW) were found more favorable for four predatory coccinellid species to attach, move and consume more aphids as compared to the P. sativum mutant line with reduced CEW . Flowering stems with high CEW of numerous other plant species (for example, species under the genera Salix, Hypenia, Eriope) often generate slippery surfaces that prevent the movement of nectar robbers, ants and other plant pests [141, 146].
Several hypotheses have been proposed and tested on the mechanisms of wax crystal inhibition of insect attachment inhibition: (i) roughness hypothesis; (ii) contamination hypothesis; (iii) fluid absorption hypothesis . Wax crystals, in general, generate a micro-rough surface on the cuticle that may prevent adhesive pads of the insects to stick, preventing them to successfully attach to the plant surface [144, 147, 148]. Contamination hypothesis proposed that detached wax crystals of the cuticular surface of some plants can adhere to the insect attachment organs (e.g., adhesive pads), contaminate those, and as such subsequent insect attachment becomes challenging and unsuccessful [147, 148, 149]. Adhesive pads of many insects secret fluids, which can also enhance wax crystal contamination to attachment organs. Fluid secretion from the adhesive pads are supposed to help insects to pursue successful attachment to the plants. However, there is evidence certain plant species have crystalline wax coverage that can absorb the fluids secreted by the adhesive pads and prevent the insects to successfully attach to the cuticle [150, 151].
The study of cuticular wax involvement in biotic stress resistance is complex with a multitude of organisms spanning insects to disease. The story is still not clear and field situations in which interactions between organisms and abiotic stresses and the role of cuticular wax needs to be evaluated. Nevertheless, certain consistencies are evident in that permeability of the cuticular layer appears to be important in pathogen invasion and wax crystals play an important role in insect intervention by the cuticular layer. These areas of research merit further investigation.
As mentioned above, abiotic stresses such as drought, extremes of temperatures, salinity, etc., cause significant losses in crop productivity. Since most of the stresses occur simultaneously, crop breeders are looking for traits contributing for multiple stress resistance. From this context, cuticular wax can serve as ideal trait. Drought stress, a major abiotic stresses in tropical regions, influences the biosynthesis and composition of cuticular wax in crops . The importance of cuticular wax in desiccation tolerance is evident that, compared to gymnosperms and angiosperms, many early extant plants such as ferns, and horsetails are more sensitive to dehydration . In crops like pea, cuticular wax load increases when subjected to drought stress . In rice, gl1-1/wsl2 and gl1-2 loss-of-function mutants with reduced wax load exhibited sensitivity to drought compared to the wild type plants [104, 153]. Drought stress is known to increase the wax content and alter composition of cuticular wax in many plants such as pea , Arabidopsis [17, 115], tobacco , alfalfa . Significant correlations between the wax content and yield, drought tolerance and water-use efficiency have been reported in different crops such as sorghum , barley , rice , and wheat [157, 158]. These reports demonstrate that less wax or non-waxy crops/genotypes are sensitive to desiccation with poor drought-tolerance compared to the crops having more cuticular wax . The existing evidences suggests cuticular wax is responsible for reducing non-stomatal transpiration by increasing cuticular resistance . The cuticular waxes also have roles in imparting resistance to salinity stress, mainly by regulating residual transpiration. A significant negative correlation observed between residual transpiration and total wax content, reports residual transpiration could be a fundamental mechanism by which plants optimize water-use efficiency under salinity stress . As discussed above, wax accumulation also correlated with high temperature resistance in plants . Leaf surface waxes help to maintain cooler canopy in sorghum under heat stress . The cuticular waxes can further help in protecting plants from high light stress . The cuticular wax has a role in protecting plants from excessive ultraviolet (UV) light and there are reports indicating that elevated UV-B radiation can affect plant cuticular wax formation [101, 159, 160]. Based on the existing information, as mentioned above, cuticular wax, can be treated as the first protective layer and an important trait contributing for both biotic and abiotic stresses.
Identification of genomic regions contributing wax traits is crucial in manipulating wax characteristics using breeding approaches. In rice, quantitative trait loci (QTL) linked to the leaf epi-cuticular layer was identified corresponding to EM15_10-ME8_4-R1394A-G2132 region on chromosome 8 . In sorghum, a crop with the ability to produce profuse amounts of EW,
With the elucidation of wax biosynthetic pathways and identification of key regulators, attempts were made in crop plants to engineer cuticle properties and to enhance stress tolerance traits. One of the early reports in engineering wax traits and thereby improved stress tolerance was from Medicago sativa (alfalfa), a forage legume. WXP1, a transcriptional regulator from Medicago truncatula, upregulated by drought, cold and ABA, was over expressed in alfalfa, which significantly increased the leaf cuticular wax load, mainly contributed by the C30 primary alcohol. The transgenic plants exhibited enhanced tolerance to drought and rapid recovery under rehydration . Over expression of SlSHN1, a close homolog of the WIN/SHN gene from Arabidopsis, in tomato using constitutive CaMV 35S promoter improved drought tolerance, with higher cuticular wax deposition on leaf epidermal tissue. The transgenic plants displayed delayed wilting, improved water status and reduced water status . MYB96, a transcriptional regulator over-expressed in Camelina, an emerging biofuel crop, which generated plants with enhanced drought tolerance. The expression levels of CsKCS2, CsKCS6, CsKCR1-1, CsKCR1-2, CsECR, and CsMAH1 were highly upregulated in the transgenic plants which resulted in a significant increase in the deposition of epicuticular waxes and total wax loads. This gives an option to cultivate the crop on marginal lands to produce renewable biofuels and bioresource . It was further demonstrated that ectopic expression of DEWAX, a negative regulator of cuticular wax biosynthesis increased tolerance to Botrytis cinerea in Camelina . A study from groundnut by over-expressing the KCS1 gene from a drought tolerant genotype improved cuticular was load and drought tolerance in a susceptible genotype . Likewise, several of such regulators have been identified from model systems as well as crop species and used for engineering crop plants to enhance stress tolerance.
In crop plants, due to the nature of combined stressors interactions, the stress effect is not always additive . While working with glossy mutants of Zea mays (gl4), an enhanced colonization of bacteria, was observed leading to more leaf blight pathogen growth compared to the wild type . The thin cuticle provided leaf blight pathogen, an easy access to nutrient and water in gl4 mutant indicating that cuticular wax thickness is a useful trait to identify plants’ resistance to combined stressors. Additionally, wax layer structure and composition are equally important in conferring defense mechanisms. As rightly pointed in Ref. , such combined studies allow us to understand the shared and specific effects of biotic and abiotic stressors.
Wild relatives and landraces have long been recognized as a source of genes for breeding major field and horticulture crops. During domestication of wheat, tomato, rice, soybean and corn, yield was the focus trait. This in turn narrowed the genetic diversity for other biotic and abiotic stressors . For example, during domestication of modern wheat, due to a phenotyping bottleneck a largely overlooked drought trait in wheat breeding program is glaucousness . Such beneficial allelic variants lost in cuticle related traits can be introgressed back by crossing an elite line with its wild relatives. Apart from genetic diversity, a mutation population (EMS or gamma irradiation) provides an alternative avenue to target crop improvement via selection of cuticle-associated trait variations . In fleshy tomatoes, a mutant line underlying for delayed fruit deterioration (DFD), is characterized for minimal transpirational water-loss and enhance post-harvest shelf life . A recent alternative for trait manipulation is CRISPR-Cas9 system which is a precise gene-editing technology. This new method accelerates the evaluation of beneficial cuticle-associated alleles in different genetic backgrounds . In similar lines, small RNA based transgenic strategy is also emerging as a molecule of choice to deal with combined biotic and abiotic resistance in crops .
There is sufficient evidence to argue that cuticle and cuticular waxes are involved in the regulation of multiple biotic and abiotic interactions. The cuticular wax can be treated as an important trait contributing for multiple stress resistance. Concerted efforts have been made to elucidate the synthesis and deposition of cuticular waxes in plants. Further analysis of the key regulatory steps involved in the formation of cuticular waxes, and also the role played by diverse types of wax components and structures in stress response is needed. This information could be incorporated in crop improvement programs (via marker assisted selection for wax genes). Since there are promising options emerging to analyze the cuticular wax trait using modern synchrotron technology  as well as now widely recognized techniques to observe ice propagation in real time across the cuticle  crop breeders have the potential to improve their efficiency of selection based on these traits. Recent progress in genomics can substantially help major field and horticulture crops to buffer the impacts of climate change. In addition, new genome-editing technologies will provide interesting tools to characterize and engineer waxes in crops. Unraveling key regulators and network partners of surface wax synthesis would aid in targeted manipulation of the trait using modern biotechnological applications. There are options to analyze the cuticular wax trait using modern non-destructive approaches. Crop breeders can use these tools to improve their efficiency of selection for the trait, and effectively pyramid the trait in elite genotypes to combat combined stresses.
RYS was supported by Agriculture and Agri-Food Canada. TR and KKT research was supported by the Agriculture Development Fund (Saskatchewan Ministry of Agriculture) and the Natural Science and Engineering Research Council (NSERC) Collaborative Research and Development program, Canada. NKN would like to acknowledge the Department of Biotechnology, Government of India, New Delhi (BT/TDS/121/SP20276/2016) and UAS Bengaluru (No. DR/Prof.(S)/RKVY/Alloc./B-44/2017-18) for the partial financial support.
Economic losses due to bovine mastitis is estimated to be $2 billion in the United States alone . Most studies showed that there is no widespread, emerging resistance among mastitis pathogens [2, 3, 4] in dairy farms. Some studies showed that the antimicrobial resistance of mastitis pathogens varies with dairy farms and bacterial species within and among dairy farms [4, 5, 6, 7, 8, 9]. However, antimicrobial resistance patterns of human pathogenic bacteria and their resistome in dairy farms might be of significant concern.
On average, starting from calving (giving birth) dairy cow is milked (in lactation) for about 300 days and then dried off (stop milking) for about 60 days before they calve again. Under the ideal dairy farming condition, a dairy cow should become pregnant within 60 days of calving, and the lactation cycle continues (Figure 1). The goal of a dry period is to give them a break from milking so that milk-producing cells regenerate, multiply, and ready for the next cycle of lactation. The incidence of intramammary infection (IMI) by bacteria is high during the early dry period and transition periods . In general, for a dairy cow, a transition period, also known as the periparturient period, is a time range from three weeks before parturition (non-milking time) until three weeks after calving (milking time). It is a transition time from non-milking to milking.
Dairy cows are susceptible to mastitis during early non-lactating (dry period) and transition periods [11, 12], especially new infection with environmental pathogens (Streptococcus spp. and coliform) are highest during the first two weeks after drying off and last two weeks before calving  compared to contagious mastitis pathogens such as S. aureus . The incidence of intramammary infection is high during the early dry period because of an absence of hygienic milking practices such as pre-milking teat washing and drying , pre- and post-milking teat dipping in antiseptic solutions [16, 17], that are known to reduce teat end colonization by bacteria and infection. An udder infected during the early dry period usually manifests clinical mastitis during the transition period  because of increased production of parturition inducing immunosuppressive hormones , negative energy balance , and physical stress during calving .
Cows are naturally protected against intramammary infections during the dry period by physical barriers such as the closure of teat opening by smooth muscle (teat sphincter) and the formation of a keratin plug, fibrous structural proteins (scleroproteins) [21, 22], in the teat canal produced by teat canal epithelium . Keratin contains a high concentration of fatty acids, such as lauric, myristic, and palmitoleic acids, which are associated with reduced susceptibility to infection and stearic, linoleic, and oleic acids that are associated with increased susceptibility to infection. Keratin also contains antibacterial proteins that can damage the cell wall of some bacteria by disrupting the osmoregulatory mechanism . However, the time of teat canal closure varies among cows. Some studies showed that 50% of teat canals were classified as closed by seven days after drying off, 45% closed over the following 50–60 days after drying off, and 5% had not closed by 90 days after dry off . Teats that do not form a plug-like keratin seal are believed to be most susceptible to infection. Infusion of long-acting antimicrobials into the udder at drying-off (dry cow therapy) has been the major management tool for the prevention of IMI during the dry period, as well as to clear IMI established during the previous lactation .
In the United States and many other countries at the end of lactation (at drying off), all cows regardless of their health status, are given an intramammary infusion of long-acting antimicrobials (blanket dry cow therapy) to prevent IMI by bacteria during the dry period [3, 25]. Because of increased concern on the use of blanket dry cow therapy for its role in driving antimicrobial resistance, selective dry cow therapy (intramammary infusion of antimicrobials into only quarters that have tendency or risk of infection) has been under investigation [26, 27]. Some recent studies showed that the use of bacteriological culture-based selective dry cow therapy at drying-off did not negatively affect cow health and performance during early lactation [26, 27]. In general, dairy farms are one of the largest users of antimicrobials including medically important antimicrobials . Some of the antimicrobials used in dairy farms include beta-lactams (penicillins, Ampicillin, oxacillin, penicillin-novobiocin), extended-spectrum beta-lactams (third-generation cephalosporins, e.g., ceftiofur), aminoglycosides (streptomycin), macrolides (erythromycin), lincosamide (pirlimycin), tetracycline, sulfonamides, and fluoroquinolones [28, 29, 30]. Antimicrobials are also heavily used in dairy farms for the treatment of cases of mastitis [3, 25, 31] and other diseases of dairy cows such as metritis, retained placenta, lameness, diarrhea, pneumonia, [32, 33, 34, 35, 36] and neonatal calf diarrhea . Over 90% of dairy farms in the US infuse all udder quarters of all cows with antimicrobial regardless of their health status [7, 25, 38]. According to dairy study in 2007 that was conducted in 17 major dairy states in the United States, 85.4% of farms use antibiotics for mastitis, 58.6% for lameness, 55.8% for diseases of the respiratory system, 52.9% for diseases of reproductive system, 25% for diarrhea or gastrointestinal infections and 6.9% for all other health problems [3, 25]. Cephalosporins were the most widely used antibiotics for the treatment of mastitis, followed by lincosamides and non-cephalosporin beta-lactam antibiotics [3, 25]. The two most commonly used antibiotics for dry cow therapy are Penicillin G/dihydrostreptomycin and cephalosporins [3, 25]. Antimicrobials were administered for the prevention and treatment of mastitis and other diseases of dairy cattle mainly through intramammary infusion and intramuscular route (USDA APHIS, 2009a). Antimicrobials infused into the mammary glands can be excreted to the environment through leakage of milk from the antimicrobial-treated udder or absorbed into the body and enter the blood circulation and biotransformed in the liver or kidney and excreted from the body through urine or feces into the environments [39, 40, 41, 42]. Similarly, antimicrobials administered through parenteral routes for the treatment of acute or peracute mastitis or other diseases of dairy cows will enter the blood circulation and biotransformed in the liver or kidney and excreted from the body through urine or feces into the environments [39, 40, 41, 42]. Therefore, both parenteral and intramammary administration of antibiotics has a significant impact on other commensals or opportunistic bacteria in the gastrointestinal tract of dairy cows and farm environments.
In addition to the use of antimicrobials for the prevention and treatment of mastitis and other diseases of dairy cattle, some farms also feed raw waste milk or pasteurized waste milk from antibiotic-treated cows to dairy calves. Feeding of raw waste milk or pasteurized waste milk from antibiotic-treated cows to calves increases pressure on gut microbes such as E. coli to became antimicrobial-resistant [43, 44, 45]. Aust et al.  showed that the proportion of antimicrobial-resistant E. coli, especially cephalosporin-resistant E. coli isolates, was significantly higher in calves fed waste milk or pasteurized waste milk from antimicrobial treated cows than calves fed bulk tank milk from non-antibiotic treated cows. However, pasteurized waste milk from cows not treated with antimicrobials is acceptable to be feed to young calves  but it is not known if pasteurization prevents the transfer of antimicrobial-resistant genes to microbes in the calve’s gut. Some studies also showed that feeding pasteurized waste milk from antimicrobial treated cows to calves increased the presence of phenotypic resistance to ampicillin, cephalothin, ceftiofur, and florfenicol in fecal E. coli compared with milk replacer-fed calves . However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves regardless of the source of milk, suggesting other driving factors for resistance development . It has been suggested that antimicrobial residues present in waste milk have a non-specific effect at a lower taxonomical level . Collectively, these non-prudent antimicrobials usage practices in dairy farms expose a large number of animals in dairy farms to antimicrobials and also increases the use of antimicrobials in dairy farms, which in turn creates intense pressure on microbes in animals’ body especially commensal and opportunistic microbes in the gastrointestinal tract and farm environments. Some of these commensal bacteria in the animal body are serious human pathogens (e.g., E. coli 0157:H7). Staphylococcus aureus is one of the pathogens with a known ability to develop antimicrobial resistance and established S. aureus infections are persistent and difficult to clear. The failure to control these infections leads to the presence of reservoirs in the dairy herd, which ultimately leads to the spread of the infection and the culling of the chronically infected cows [46, 47].
Monitoring antimicrobial resistance patterns of bacterial isolates from cases of mastitis is important for treatment decisions and proper design of mitigation measures. It also helps to determine emergence, persistence, and potential risk of the spread of antimicrobial-resistant bacteria and resistome to human, animal, and environment [48, 49]. The prudent use of antimicrobials in dairy farms reduce emergence, persistence, and spread of antimicrobial-resistant bacteria and resistome from dairy farms to human, animal, and environment.
Most studies showed that there is no widespread, emerging resistance among mastitis pathogens [2, 3, 4] in dairy farms. However, dairy farms may serve as a source of antimicrobial-resistant human pathogenic bacteria. Extensive use of third-generation cephalosporins (3GCs) in dairy cattle for the prevention and treatment of mastitis [3, 25, 28] and other diseases of dairy cattle [31, 32] can result in the carriage of extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL Ent) [50, 51]. Third- and fourth-generation cephalosporins are commonly used for the treatment of invasive Gram-negative bacterial infections in humans [52, 53, 54]. In 2017, there were an estimated 197,400 cases of ESBL Ent among hospitalized patients and 9100 estimated deaths in the US alone . Among Enterobacteriaceae, Escherichia coli (E. coli) is the most common bacteria that reside in the gut as normal microflora or opportunist pathogen of animals and humans. However, certain pathogenic strains can cause diseases such as mastitis in cattle, neonatal calf diarrhea in calves and hemorrhagic enteritis, and more life-threatening conditions such as hemolytic uremic syndrome and urinary tract infections in humans. New strains of multi-drug resistant foodborne pathogens that produce extended-spectrum beta-lactamases that inactivate nearly all beta-lactam antibiotics have been reported . Ceftiofur is the most common 3GC used in dairy cattle operations . The 3GCs are also critically important antibiotics for the treatment of serious infections caused by Enterobacteriaceae such as Escherichia coli (E. coli) and Salmonella spp. in humans [57, 58]. The use of structurally and chemically similar antibiotics in dairy cattle production and human medicine may lead to co-resistance or cross-resistance [52, 53, 54]. Some of the species of Gram-negative environmental mastitis pathogens, such as E. coli, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas spp., Enterobacter spp. are the greatest threat to human health due to the emergence of strains that are resistant to all or most available antimicrobials [59, 60].
The resistance of Enterobacteriaceae to 3GC is mainly mediated by the production of extended-spectrum beta-lactamase enzymes (ESBLs) that breakdown 3GC . E. coli is one of the most frequently isolated Enterobacteriaceae carrying ESBL genes (blaCTX-M, blaSHV, blaTEM, and blaOXA) families [62, 63, 64]. These ESBL genes are usually carried on mobile plasmids along with other resistance genes such as tetracycline, quinolones, and aminoglycosides. E. coli resides in the gastrointestinal tract of cattle as normal or opportunistic microflora, but some strains (for e.g., 0157:H7) cause serious infection in humans , indicating that cattle could serve as a reservoir of ESBLs producing E. coli (ESBLs E. coli) for human.
In the US, the occurrence of ESBLs E. coli in the dairy cattle was reported a decade ago from Ohio  and few previous studies reported the occurrence and an increase in the trend of ESBLs E. coli in the dairy cattle production system [52, 53, 65, 66, 67]. However, recent studies increasingly showed the rise of ESBLs E. coli in the cattle [51, 52, 65, 67]. Similarly, reports from the Center for Disease Control (CDC) showed a continuous increase in the number of community-associated human infections caused by ESBLs-producing Enterobacteriaceae . This CDC report showed a 9% average annual increase in the number of hospitalized patients from ESBLs pathogens in six consecutive years (from 2012 to 2017). As a result, the human health sector tends to blame dairy farms that routinely use the 3GC for the rise of ESBLs pathogens such as E. coli [55, 68]. However, despite the general believe of possibility of transmission of antimicrobial-resistant bacteria from dairy farms to humans directly through contact or indirectly through food chain, there was no clear evidence-based data that showed the spread of antimicrobial-resistant bacteria from the dairy production system to humans. The opinion of the scientific community on the factors that drive the emergence and spread of antimicrobial-resistant bacteria also varies . Transmission of an antimicrobial-resistant pathogen to humans could occur if contaminated unpasteurized milk and/or undercooked meat from culled dairy cows due to chronic mastitis is consumed . So it is crucial to pasteurize milk or cook meat properly to reduce the risk of infection by antimicrobial-resistant bacteria . It is not known, if pasteurization or proper cooking prevents the transfer of resistant genes from milk or meat to commensal or opportunistic bacteria in the human gastrointestinal tract (GIT), or the GIT of calves fed pasteurized waste milk. Mechanisms of antibiotic resistance gene transfer from resistant to susceptible bacteria are not well known, and killing resistant pathogens alone may not be good enough to prevent the transfer of the resistance gene. Non-prudent use of antimicrobials in dairy farms increases selection pressure, which could result in the emergence, persistence, and horizontal transfer of antimicrobial-resistant determinants from resistant to non-resistant bacteria. Bacteria exchange resistance genes through mobile genetic elements such as plasmids, bacteriophages, pathogenicity islands, and these genes may ultimately enter bacteria pathogenic to humans or commensal or opportunistic bacterial pathogens. The prudent use of antimicrobials in dairy farms requires identification of the pathogen causing mastitis, determining the susceptibility/resistance of the pathogen, and proper dose, duration, and frequency of treatment to ensure effective concentrations of the antibiotic to eliminate the pathogen.
Despite decades of research to develop effective vaccines against major bacterial bovine mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, and E. coli, the effective intramammary immune mechanism is still poorly understood, perpetuating reliance on antibiotic therapies to control mastitis in dairy cows. Dependence on antimicrobials is not sustainable because of their limited efficacy [46, 47] and increased risk of emergence of antimicrobial-resistant bacteria that pose serious public health threats [4, 72, 73, 74]. Neither of the two currently available commercial Bacterin vaccines against S. aureus (Table 1), Lysigin® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) in the USA and Startvac® (Hipra, Girona, Spain) in Europe and other countries, confer protection from new intramammary infection under field trials as well as under controlled experimental challenge studies [75, 76, 77, 78, 79, 80, 81].
|Mastitis Pathogen||Vaccine||Vaccine component||Protective effect||Reference|
|S. aureus||Lysigin®||Bacterin: Somatic antigen containing phage types I, II, III, IV with different strains of S. aureus||Reduced SCC, clinical mastitis, and chronic IMI||[193, 194, 195]|
|“||“||Field-based studies concluded no such effect||[80, 81, 111, 114, 196]|
|Startvac®||Bacterin: E. coli J5 and S. aureus CP type 8 with SAAC||Decreased duration of IMI, transmissibility of S. aureus, coliforms, and CNS|||
|“||“||Use of the vaccine was not associated with a decrease in mastitis|||
|Bestvac® Vs Startvac||herd-specific autologous vaccine compared with Startvac®||Both vaccines decreased herd prevalence of S. aureus mastitis but no other differences in terms of improvement of udder health|||
|Whole-cell lysate||Bacterin encapsulated in biodegradable microspheres||Induced antibodies that were more opsonic for neutrophils and inhibited adhesion to mammary epithelium.|||
|Whole-cell lysate from two strains||Bacterin from two strains (α and α + β hemolytic) plus supernatants from non-hemolytic strain||Vaccinated cows had 70% protection from infection compared to less than 10% protection in control cows|||
|MASTIVAC I||Whole-cell lysate||Improved udder health in addition to specific protection against S. aureus infection|||
|Live pathogenic S. aureus through IM route||Live pathogenic S. aureus||Induce activation of immune cells in mammary gland and blood|||
|Fibronectin binding protein and clumping factor A||DNA primed and protein boosted||Induced cellular and humoral immune responses that provide partial protection against S. aureus|||
|Protein A of S. aureus with the green fluorescent protein||DNA||Induced humoral and cellular immune responses|||
|Plasmid encoding bacterial antigen β-gal||DNA||Induced humoral and cellular immune responses|||
|Polyvalent S. aureus Bacterin||Bacterin||Eliminated some cases of chronic intramammary S. aureus infections|||
|Lysigin® with three-isolates based experimental Bacterin||Bacterin||Lysigin reduced the clinical severity and duration of clinical disease. None of the experimental Bacterins has significant effects|||
|Polyvalent S. aureus Bacterin||Bacterin + antibiotic therapy||S. aureus intramammary infection cure rate increased|||
|Whole-cell lysate||Whole-cell trivalent vaccine containing CP types 5, 8 and 336 with FIA or Alum adjuvants||Elicited antibody responses specific to the 3 capsular polysaccharides|||
|CP conjugated to a protein and incorporated in polymicrospheres and emulsified in FIA||CP types 5, 8 and 336||Cows in both groups produced increased concentrations of IgG1, IgG2 antibodies, hyperimmune sera from immunized cows increased phagocytosis, decreased bacterial adherence to epithelial cells|||
|Polysaccharide-protein conjugates in FIA||Polysaccharide-protein conjugate|
|SASP or SCSP||Surface proteins||Induced partial protection|||
|Vaccination with Efb and LukM||Induced increased titers in serum and milk|||
|Inactivated Bacterin||Bacterin||Partial protection|||
|UBAC®||Extract from biofilm-forming strains of S. uberis||Reduce clinical signs, bacterial count, temperature, daily milk yield losses and increased the number of quarters with isolation and somatic cell count <200,000 cells/mL of milk|||
|Killed S. uberis cells||Bacterin||Reduced numbers of homologous S. uberis in milk|||
|Killed bacterial cells||Bacterin of S. uberis and S. agalactiae||Parenteral vaccination has no effect on streptococcal mastitis||[200, 201]|
|Live S. uberis/ cutaneous route||Live S. uberis||Some protective effect only on the homologous strain|||
|GapC or chimeric CAMP factor||Protein||Reduction in inflammation|||
|E. coli J5|
|Bacterin||Reduce bacterial counts in milk, duration of IMI and resulted in fewer clinical symptoms||[82, 83, 188, 189, 190]|
There are four commercial vaccines against E. coli mastitis which include 1) the Eviracor®J5 (Zoetis, Kalamazoo, MI), [82, 83], 2) Mastiguard®, 3) J-VAC® (Merial-Boehringer Ingelheim vet medical, Inc., Duluth, GA) and 4) ENDOVAC-Bovi® (IMMVAC) (Endovac Animal Health, Columbia, MO) (Table 1). The Endovac-bovi® is a cross-protective vaccine made of genetically engineered R/17 mutant strain of Salmonella typhimurium and the core somatic antigen mutant J-5 strain of E. coli combined with an immune-potentiating adjuvant (IMMUNEPlus®). Endovac-bovi significantly reduces diseases caused by Gram-negative bacteria producing various endotoxins and protects against E. coli mastitis and other endotoxin-mediated diseases caused by E. coli, Salmonella, Pasteurella multocida, and Mannheimia hemolytica. The UBAC® (Hipra, Amir, Spain)  is a recently developed vaccine against S. uberis mastitis with label claim of partial reduction in clinical severity of S. uberis mastitis.
Intramammary immunity can be induced locally in the mammary gland or systemically in the body and cross from the body into the mammary glands. Mammary gland pathogen that enters through teat opening interact with host innate defense system primarily with macrophages in the mammary gland. Macrophages recognize invading pathogens through its pattern recognition receptors (PRR) which binds to pathogen associated molecular patterns (PAMPs) and engulf and break down the foreign pathogen into small peptides and load on to MHC-II molecules move to the supramammary lymph nodes and display on its surface to the T cells. Naïve T cells bind with peptide on MHC-II molecule through its T- cell receptor and become activated and start secreting cytokines, which further stimulate B-cells to produce antibodies. Antibody produced by B-cells released into the blood circulation and depending on type of antibody may be released to the site of infection (e.g., IgG) and opsonize the infecting pathogen and subject them to destruction by opsonophagocytic mechanisms. Antibodies may also remain on mucosal surfaces (e.g., IgA) and bind to invading pathogens and prevent them from binding to host cells or tissue and thereby prevent colonization and infection.
Intramammary infection (IMI) leads to increased somatic cell count in the milk or mammary secretion. Somatic cells are mainly white blood cells such as granulocytes (neutrophils, eosinophils, and basophils), monocytes or macrophages, and lymphocytes, which are recruited to the mammary glands in response to mammary gland infection to fight off infection. A small proportion of mammary epithelial cells that produce milk are also shed through milk and are included in the somatic cell count. So, somatic cells are white blood cells and mammary epithelial cells. Milk somatic cell count (SCC) increases when there is mammary gland infection (IMI) because of an inflammatory response to clear infection. In general, SCC is also an indicator of milk quality [85, 86, 87, 88, 89] because if there are few mammary pathogenic bacteria in the gland, the inflammatory response is less, and somatic cells recruitment into the gland is also low and vice versa. Bulk tank milk (BTM) is milk collected from all lactating dairy cows in a farm into a tank or multiple tanks. So BTSCC is somatic cell counts obtained from milk sample collected from a tank.
Intramammary infection may progress to clinical or subclinical mastitis . Clinically infected udder usually treated with antimicrobial, whereas subclinically infected udder may not be diagnosed immediately and treated but remained infected and shedding bacteria through milk throughout lactation. The proportion of cure following treatment of mastitis varies and the variation in cure rate is multi-factorial including cow factors (age or parity number, stage of lactation, and duration of infection, etc.), management factors (detection and diagnosis of infection and time from detection to treatment, availability of balanced nutrition, sanitation, etc.), factors related to antimicrobial use patterns (type, dose, route, frequency, and duration), and pathogen factors (type, species, number, pathogenicity or virulence, resistance to antimicrobial, etc.) [46, 91].
The dilution of effector humoral immune responses by large volume of milk coupled with the ability of mastitis causing bacteria to develop resistance to antimicrobials makes the control of mastitis very difficult. Therefore, the development of an alternative preventive tool such as a vaccine, which can overcome these limitations, has been a crucial focus of current research to decrease not only the incidence of mastitis but also the use of antimicrobials in dairy cattle farms. Most vaccination strategies against mastitis have focused on the enhancement of humoral immunity. Development of vaccines that induce an effective cellular immune response in the mammary gland has not been well investigated. The ability to induce cellular immunity, especially neutrophil activation and recruitment into the mammary gland, is one of the key strategies in the control of mastitis, but the magnitude and duration of increased cellular recruitment into the mammary gland leads to a high number of somatic cells and poor-quality milk. So, effective balanced humoral and cellular immunity that clear intramammary infection in a short period of time is required. Several vaccine studies were conducted over the years under controlled experimental and field trials. The major bacterial bovine mastitis pathogens that have been targeted for vaccine development are S. aureus, S. uberis, and E. coli . Most of these experimental and some commercial vaccines are Bacterins which are inactivated whole organism, and some vaccines contained subunits of the organism such as surface proteins , toxins, or polysaccharides.
Staphylococcus aureus is one of the most common contagious mastitis pathogens, with an estimated incidence rate ranging from 43–74% [25, 38, 56, 94, 95]. Staphylococcus chromogenes is another increasingly reported coagulase-negative Staphylococcus species with an estimated quarter incidence rate of 42.7% characterized by high somatic cell counts [96, 97, 98, 99, 100, 101, 102]. In a study on conventional and organic Canadian dairy farms, coagulase-negative Staphylococcus species were found in 20% of the clinical samples . Recently, mastitis caused by coagulase-negative Staphylococcus species increasingly became more problematic in dairy herds [99, 101, 104, 105].
Several staphylococcal vaccine efficacy trials showed that vaccination with Bacterin vaccines induced increased antibody titers in the serum and milk that are associated with partial protection [75, 76, 77, 80, 106, 107, 108, 109, 110, 111, 112] or no protection at all [78, 79, 81]. However, effective intramammary immune mechanisms against staphylococcal mastitis is still poorly understood. None of the commercially available Bacterin vaccines protects new intramammary infection [75, 77, 80, 81]. Dependence on antibiotics for the prevention and treatment of mastitis is not sustainable because of limited success [46, 47] and the emergence of antimicrobial-resistant bacteria that are major threat to human and animal health [72, 73, 74].
Despite several mastitis vaccine trials conducted against S. aureus mastitis [75, 77, 80, 107, 108, 109, 110, 111, 113, 114, 115, 116, 117], all field trials have either been unsuccessful or had limited success. There are two commercial vaccines for Staphylococcus aureus mastitis on the market, Lysigin® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) in the United States and Startvac® (Hipra S.A, Girona, Spain) in Europe and Canada . None of these vaccines confer protection under field trials as well as under controlled experimental studies [75, 77, 80, 81]. Several field trials and controlled experimental studies have been conducted testing the efficacy of Lysigin® and Startvac®, and results from those studies have shown some interesting results, namely a reduced incidence, severity, and duration of mastitis in vaccinated cows compared to non-vaccinated control cows [75, 76, 77]. Contrary to these observations, other studies failed to find an effect on improving udder health or showed no difference between vaccinated and non-vaccinated control cows [78, 79]. None of these Bacterin-based vaccines prevents new S. aureus IMI [75, 77, 80, 81]. Differences found in these studies are mainly due to methodological differences (vaccination schedule, route of vaccination, challenge model, herd size, time of lactation, etc.) in testing the efficacy of these vaccines. It is critically important to have a good infection model that mimics natural infection and a model that has 100% efficacy in causing infection. Without a good challenge model, the results from vaccine efficacy will be inaccurate.
The Startvac® (Hipra, Girona, Spain) is the commercially available vaccine in Europe and is a polyvalent vaccine that contains E. coli J5 and S. aureus strain SP140 . In a field trial, Freick et al.  compared the efficacy of Startvac® with Bestvac® (IDT, Dessau-Rosslau, Germany) another herd-specific autologous commercial vaccine in a dairy herd with a high prevalence of S. aureus and found that the herd prevalence of S. aureus mastitis was lower in the Startvac® and Bestvac® vaccinated cows compared to the control cows. However, there were no other differences in terms of improvement of udder health. These authors  concluded that vaccination with Startvac® and Bestvac® did not improve udder health. In another field efficacy study on Startvac® in the UK, Bradley et al.  found that Startvac® vaccinated cows had clinical mastitis with reduced severity and higher milk production compared to non-vaccinated control cows .
Similarly, Schukken et al.  evaluated effect of Startvac® on the development of new IMI and the duration of infections caused by S. aureus and CNS. These authors  found that vaccinated cows had decreased incidence rate and a shorter duration of S. aureus and CNS mastitis. Piepers et al. , also tested the efficacy of Startvac® through vaccination and subsequent challenge with a heterologous killed S. aureus strain and found that the inflammatory response in the vaccinated cows was less severe compared to the control cows. These authors  suggested that Startvac® elicited a strong Th2 immune response against S. aureus in vaccinated cows and was more effective at clearing bacteria compared to the control cows. Contrary to these observations, Landin et al. , evaluated the effects of Startvac® on milk production, udder health, and survival on two Swedish dairy herds with S. aureus mastitis problems and found no significant differences between the Startvac® vaccinated and non-vaccinated control cows on the health parameters they evaluated.
An experimental S. aureus vaccine made up of a combination of plasmids encoding fibronectin-binding motifs of fibronectin-binding protein (FnBP) and clumping factor A (ClfA), and plasmid encoding bovine granulocyte-macrophage-colony stimulatory factor, was used as a vaccine with a subsequent challenge with bacteria to test its protective effects . These authors (Shkreta et al. 2004) found that their experimental vaccine-induced immune responses in the heifers that were partially protective upon experimental challenge . Another controlled experimental vaccine efficacy study was conducted on the slime associated antigenic complex (SAAC) which is an extracellular component of Staphylococcus aureus, as vaccine antigen in which one group of cows were vaccinated with a vaccine containing a low amount of SAAC and another group with a high amount of SAAC and the unvaccinated group served as a control . Upon intramammary infusion (challenge) with S. aureus, no difference in the occurrence of mastitis among all three groups despite the fact that the vaccine with high SAAC content induced higher production of antibodies compared to the vaccine with a low amount of SAAC . Similarly, Pellegrino et al. , vaccinated dairy cows with an avirulent mutant strain of S. aureus and subsequently challenged with S. aureus 20 days after the second vaccination which resulted in no significant differences in the number of somatic cell count (SCC) or number of bacteria shedding through milk despite increased IgG antibody titer in the vaccinated cows compared to the control cows.
Some of the constraints affecting the successful development of effective mastitis vaccines are strain variation, the presence of exopolysaccharide (capsule, slime, biofilm) layer in most pathogenic strains of bacteria (Staph. aureus, Strep. uberis) which does not allow recognition of antibody-coated bacteria by phagocytic cells, dilution of immune effectors by milk [121, 122], the interaction between milk components and immune effectors  that reduce their effectiveness, and the ability of most mastitis-causing bacteria to attach and internalize into mammary epithelial cells. Furthermore, evaluation of mastitis vaccines is complicated by the absence of uniform challenge study models, and lack of uniform route(s) of vaccination, time of vaccination, adjuvants, and challenge dose. There is an increasing need for development of better vaccines that overcome these problems. Most mastitis vaccines are killed whole bacterial cells (Bacterin) vaccines [75, 77, 80, 108, 109, 110, 111, 113, 114, 115, 116, 117, 124] that are difficult to improve because of difficulty to specifically identify an immunogenic component that induced partial or some protective effect. In this regard, some of the current efforts to use a mixture of purified surface proteins as vaccine antigens  to induce immunity than killed whole bacterial cells (Bacterin) is encouraging. A better understanding of natural and acquired immunological defenses of the mammary gland coupled with detailed knowledge of the pathogenesis of each mammary pathogen should lead to the development of improved methods of reducing the incidence of mastitis in dairy cows.
S. uberis is ubiquitous in the cow’s environment accounting for a significant number of mastitis cases. It is found on-farm in water, soil, plant material, bedding, flies, hay, and feces . As such, S. uberis is remarkably adaptable, affecting lactating and dry cows, heifers, and multiparous cows, causing clinical or subclinical mastitis, and even being responsible for persistent colonization without an elevation in the somatic cell count [126, 127]. It has been described as an environmental pathogen [128, 129, 130, 131] with potential as a contagious pathogen [126, 127, 132]. S. uberis has ability to persist within the mammary gland which lead to chronic mastitis that is difficult to treat . Coliform bacteria are a major cause of clinical mastitis [134, 135]. A vaccine that prevents S. uberis mastitis is not available, control measures are limited to the implementation of good management practices. Recently vaccine efficacy trial with extract of biofilm-forming strains of S. uberis (UBAC®) (Hipra, Amir, Spain), was reported to reduce clinical severity . It is not clear what kind of adative immunity is induced by UBAC® S. uberis vaccine  and it only conferred partial reduction in clinical severity of mastitis. Multiple intramammary vaccinations of dairy cows with killed S. uberis cells resulted in the complete protection from experimental infection with the homologous strain . Similarly, subcutaneous vaccination of dairy cows with live S. uberis followed by intramammary booster vaccination with S. uberis cell surface extract protected against challenge with the homologous strain but was less effective against a heterologous strain . Vaccination with S. uberis glyceraldehyde phosphate dehydrogenase C (GapC) protein induced immune responses that confer a significant reduction in inflammation post-challenge [138, 139]. The pauA is a plasminogen activator and also binds active protease plasmin . It has been postulated that acquisition of plasmin may promote invasion . Vaccination of dairy cows with PauA induced increased antibody titers that conferred reduction in clinical severity . However, mutation of pauA did not alter ability to grow in milk or to infect lactating bovine mammary glands. It appears that the ability to activate plasminogen through PauA does not play a major role in pathogenesis of S. uberis to either grow in milk or infect bovine mammary gland .
S. uberis expresses several surface associated proteins such as S. uberis adhesion molecule (SUAM) and extracellular matrix binding proteins, which allow it to adhere to and internalize into mammary epithelial cells, successfully inducing IMI [144, 145, 146]. The S. uberis adhesion molecule (SUAM) plays a central role in the adherence of S. uberis to mammary epithelial cells [147, 148, 149, 150]. Vaccination of dairy cows with SUAM induced strong immune resposes in vaccinated cows . The immune serum from SUAM vaccinated cows prevented S. uberis adhesion and invasion into mammary epithelial cells in vitro . In vivo infusion of mammary quarters of dairy cows with S. uberis pre-incubated with immune-serum from SUAM vaccinated cows reduced clinical severity . The SUAM gene deletion mutant strain is less pathogenic to mammary epithelial cells  and to dairy cows . Controlled experimental efficacy studies using SUAM as vaccine antigen to control S. uberis mastitis showed that SUAM is immunogenic but the induced immunity was not protective. Following experimental IMI challenge with S. uberis, clinical signs emerged at about 48 h, along with increased levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-8 in milk at 60 h post-infection . Adaptive immune response cytokines such as IFN-γ promotes a cell-mediated immune response by enhancing functions such as macrophage bacterial killing, antigen presentation, cytotoxic T cell activation, and increased IgG2 levels. The IL-4 expression is associated with the antibody-mediated response, which is generally linked to parasite resistance, allergic reactions, and increased levels of IgG1 [155, 156]. This partial protection by the SUSP vaccine can be improved with dose optimization, appropriate adjuvant, route of injection, and timing of vaccination.
In conclusion, it is clear that Bacterin vaccines have some protective effect against homologous strains, and single surface protein is not effective. Therefore; use of multiple surface proteins may induce better immunity that prevents clinical disease and production losses.
Coliform bacteria are a major cause of clinical mastitis [134, 135]. Coliforms include the genera Escherichia, Klebsiella, and Enterobacter . Eighty to ninety percent of coliform intramammary infection (IMI) develop clinical mastitis, and 10% will be severe and could lead to death . E. coli usually infects the mammary glands during the dry period and progresses to inflammation and clinical mastitis during the early lactation with local and sometimes severe systemic clinical manifestations.
Iron is an essential nutrient for the growth of coliforms . However, free iron is limited in the bovine milk because most iron is bound to citrate and to a lesser extent to lactoferrin, transferrin, xanthine oxidase, and some caseins  and maintained at concentrations below levels required to support coliform growth . To overcome this limited iron source, coliforms express multiple iron transport systems , which include synthesis of siderophores (e.g., enterobactin, aerobactin, ferrichrome) that bind iron with high affinity , the expression of iron-regulated outer membrane proteins (IROMP) that binds to ferric siderophore complexes to transport into bacterial cell and enzymes to utlize the chelated iron . The siderophores are too large (600 to 1200 Da) to pass through the porin channels of the bacterial outer membrane [163, 164]. Therefore, the siderophores require specific IROMP to enable their passage across the bacterial outer membrane into the periplasm [165, 166]. The enterobactin is a siderophore with the highest affinity for iron, and it is produced by most pathogenic E. coli and Klebsiella spp. [167, 168, 169]. The aerobactin is another siderophore that was detected in only 12% of E. coli isolated from mastitis cases . Enterobactin is the primary siderophore of Escherichia coli and many other Gram-negative bacteria . Coliform bacteria also developed the ability to take up iron directly from naturally occurring organic iron-binding acids, including citrate [161, 172]. The citrate iron uptake system requires ferric dicitrate for induction . More than 0.1 mM citrate is required for the induction of this system under iron-restricted conditions . The ferric citrate transport system is the major iron acquisition system utilized by E. coli  to grow in the mammary gland. The mammary gland is an iron-restricted environment, and bovine milk contains approximately 7 mM citrate  which is ideal for induction of ferric citrate transport sytem.
Ferric enterobactin receptor, FepA, is an 81 kDa iron regulated outer membrane protein (IROMP), that binds to ferric enterobactin complex to transoport iron into the bacterial cell [174, 175]. Vaccination of dairy cows with FepA elicited an increased immunological response in serum and milk . Bovine IgG directed against FepA inhibited the growth of coliform bacteria by interfering with the binding of the ferric enterobactin complex . Ferric citrate receptor, FecA, is an 80.5-kDa IROMP that is responsible for the binding of ferric dicitrate  and transport into the bacterial cell. The FecA, is conserved among coliforms isolated from cases of naturally occurring mastitis . The iron-regulated outer membrane proteins, FepA and FecA are ideal vaccine candidates because they are surface exposed, antigenic, and conserved among isolates from IMI.
Immunization of dairy cows with FepA induced significantly higher serum and whey anti-FepA IgG titers than in E. coli J5 vaccinates . Results of in vitro growth inhibition studies demonstrated that antibody specific for blocking ferric enterobactin-binding site (anti-FepA) inhibited the growth of E. coli in vitro . Cows immunized with FecA did have increased antibody titers in serum and mammary secretions compared with E. coli J5 immunization and unimmunized control cows [181, 182]. Antibody purified from colostrum inhibited the growth of E. coli when cultured in synthetic media modified to induce FecA expression . Despite their antigenicity, the use of either FepA or FecA alone were not sufficient to prevent mastitis. The FecA and FepA are antigenically distinct .
Intramammary infection with E. coli induced expression and release of pro-inflammatory cytokines such as TNF-alpha, IL-8, IL-6, and IL-1 [183, 184]. Recently it has been shown with mouse mastitis models that IL-17A and Th17 cells are instrumental in the defense against E. coli IMI [185, 186]. However, the role of IL-17 in bovine E. coli mastitis is not well defined. Results of a recent vaccine efficacy study against E. coli mastitis suggested that cell-mediated immune response has more protective effect than humoral response . The cytokine signaling pathways that lead to efficient bacterial clearance is not clearly defined.
The four coliform vaccines which include 1) J-5 Bacterin® (Zoetis, Kalamazoo, MI) [82, 83], 2) Mastiguard®, 3) J Vac® (Merial-Boehringer Ingelheim vet medical, Inc., Duluth, GA) and 4) Endovac-bovi® (IMMVAC) (Endovac Animal Health, Columbia, MO). Of the four coliform vaccines, J-5 Bacterin® and Mastiguard® are believed to have the same component, which is J5 Bacterin. The J Vac® is a different bacterin-toxoid. The Endovac-Bovi® contains mutant Salmonella typhimurium bacterin toxoid. All coliform mastitis vaccine formulations use gram-negative core antigens to produce non-specific immunity directed against endotoxin (LPS) . The efficacy of these vaccines has been demonstrated in both experimental challenge trials and field trials in commercial dairy herds [188, 189, 190]. The principle of these bacterins is based upon their ability to stimulate the production of antibodies directed against common core antigens that gram-negative bacteria share. These vaccines are considered efficacious even though the rate of intramammary infection is not significantly reduced in vaccinated animals because they significantly reduce the clinical effects of the infection. Experimental challenge studies have demonstrated that J5 vaccines are able to reduce bacterial counts in milk and result in fewer clinical symptoms . Vaccinated cows may become infected with gram-negative mastitis pathogens at the same rate as control animals but have a lower rate of development of clinical mastitis , reduced the duration of IMI , reduced production, culling, and death losses [191, 192].
There is an increasing need for the development of effective vaccines against major bacterial bovine mastitis pathogens. A better understanding of the natural and acquired immunological defenses of the mammary gland coupled with detailed knowledge of the pathogenesis of each mammary pathogen should lead to the development of improved methods of reducing the incidence of mastitis in dairy cows (Table 1).