a proteomics perspective from animal welfare to food safety

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156 Current Protein and Peptide Science, 2014, 15, 156-168

A Proteomics Perspective: From Animal Welfare to Food Safety

Anna Bassols1, Romana Turk

2 and Paola Roncada

3,4,*

1Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona Cer-

danyola del Vallès, Barcelona, Spain; 2Faculty of Veterinary Medicine, University of Zagreb, Department of Patho-

physiology, Zagreb, Croatia; 3Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano;

4Dipartimento di Scienze

Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy

Abstract: A fundamental issue of farm animal welfare is to keep animals clinically healthy, without disease or stress, par-

ticularly in intensive breeding, in order to produce safe and quality food. This issue is highly relevant for the food industry

worldwide as they are directly linked to public health and welfare. The aim of this review is to explore how proteomics

can assess and improve the knowledge useful for the strategic management of products of animal origin. Useful indica-

tions are provided about the latest proteomics tools for the development of novel biotechnologies serving the public

health. The multivariate proteomics approach provides the bases for the discovery of biomarkers useful to investigate ad-

aptation syndromes and oxidative stress. These two responses represent the milestones for the study of animal welfare.

Moreover their implementation in the characterization and standardization of raw materials, process development, and

quality and safety control of the final product of animal origin represents the current frontier in official surveillance and

tests development.

Keywords: Animal welfare, foodborne pathogens, food safety, immunoresponse, proteomics, stress.

1. INTRODUCTION

Animal welfare is a hallmark to assess food quality and

safety. The safety of food chain is indirectly influenced by animal welfare. This is given by the strict link that occurs between animal welfare, animal health and foodborne patho-gens. Stress factors and the lack of healthy conditions for

animal welfare may lead animals to increase their suscepti-bility to diseases. This could represent a serious issue for consumers who may in turn be exposed to common food borne pathogens such as Salmonella, Campylobacter and E.

coli. If the general management of a breeder is correct under hygienic conditions, the key point influencing animal wel-fare is stress. Proteomics represents an innovative biotechno-logical approach to act and investigate at different levels of

the food chain. The implementation of the new biotechno-logical tools derived from proteomics represents an impor-tant challenge for the development of responsible biotech-nology serving the public health by providing advanced ana-

lytical methods. The aim of this review is to provide the fun-damental concepts of the potential application of proteomics in different areas of food quality and safety. In particular the power of proteomic tools to investigate how animal welfare

can influence food production will be highlighted.

Proteomics offers the possibility of a global overview of the whole organism adaptation response to stress conditions and provides information on biomarkers that may be signifi-cant indicators of animal welfare based on a molecular

*Address correspondence to this author at the Dipartimento di Scienze Vet-

erinarie e Sanità Pubblica, Università degli Studi di Milano, Via Celoria 10,

20133 Milano, Italy; Tel: +390250318138;

E-mails: [email protected] and [email protected]

analytical response. Proteins are major targets for stress-associated post-translational modifications, such as oxida-tion, given their high overall abundance in biological sys-tems and because they are primarily responsible for most functional processes within the cells. In a condition of in-creased oxidative status of the organism, a proteomic ap-proach allows to detect and identify changes in the concen-tration of enzymes involved in redox metabolism as well as in the level of oxidised proteins. Oxidative modifications in the protein structure can provide a typical fingerprint such as carbonylation of amino acids, detectable through proteomic tools [1-3]. Farm animals are subjected to physiological, pathological and environmental conditions that can affect health as well as characteristics of the final product (meat, milk, etc.) and safety issues and food quality aspects (meat tenderness, protein composition, etc.). Since animals are many times bred in crowded farms, this leads to a highly challenging and adverse environment and, sometimes, to multiple risks for the onset of infectious diseases with a high risk of propagation. Problems concerning welfare could be due to excessive crowding of the animals, but also to mixing of the animals leading to a disruption of their hierarchical social structures (specially with pigs), transport to the slaughterhouse in long and uncomfortable travels, etc [4, 5].

Furthermore, the genetic selection in the direction of im-proved food quantity and quality produced extreme pheno-types with increasing health problems (for example, high incidence of bovine mastitis associated to extreme milk yields, or colostrum deprivation in hyperprolific sows). Both intensive animal production and genetic selection results in an overall decrease of animal welfare. For this reason it is more and more necessary to know the mechanisms of patho-genesis of the most frequently occurring diseases in farm

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A Proteomics Perspective: From Animal Welfare to Food Safety Current Protein and Peptide Science, 2014, Vol. 15, No. 2 157

animals to control welfare and safety issues. In this context, proteomics has an important role for the discovery of bio-markers to monitor health and welfare and in the study of diseases and the production of new vaccines [6-8]. The over-all final aim is to optimize a sustainable equilibrium between productivity, product quality and animal welfare.

Fig. (1). Factors that can direcly or indirectly influence food safety.

Fig. (2). How proteomics can act at different levels from farm to

fork.

2. STRESS AND ITS IMPACT ON FARM ANIMAL

WELFARE

Different stressors, such as transport, housing and tem-

perature conditions, feed deprivation before slaughtering, physiological conditions like pregnancy and lactation, patho-

gens etc. can trigger a nonspecific response needed to re-

establish homeostasis of the body. Moreover, during space allocation, animals are exposed to a wide range of stimuli,

including increased human contact, vibration during trans-

port, unknown environments and food and water deprivation.

Stress is defined as the unspecific response of the body to different exogenous or external adverse environment [9].

The stress response provokes a range of modifications usu-

ally called general adaptation syndrome. Moreover, chronic stress may result in pathological states and lead to distur-

bances of health, growth and fertility thus impairing the effi-

ciency of animal production[10]. Chronic stress, in particu-lar, suppresses the immune system and increases the suscep-

tibility to infections, pathogen shedding, and carcass con-

tamination [11].

Welfare problems may cause great economic losses and

are important for ethical reasons and public opinion. In this context, there is an increasing interest in the measurement of

stress as an indicator of animal welfare, nutritional status and

disease. Behavioral and physiological markers are commonly used, but they may increase by other causes [12, 13]. Indica-

tors of nutritional stress include mainly non-esterified fatty

acids (NEFAs) and 3-hydroxybutyrate. Acute phase proteins (APPs), such as Pig-MAP and haptoglobin, are recognized

markers of inflammation [14, 15] and have also been pro-

posed as indicators of stress in cattle and pigs [16, 17]. Nev-ertheless, objective laboratorial criteria to evaluate animal

stress are still lacking and proteomics may be useful to

achieve this goal.

2.1. Impact of Stress on Farm Animal Production

Stress results in the release of a number of different hor-mones, neurotransmitters, cytokines and other factors into the circulation or tissues. Particularly, increased levels of glucocorticoids inhibit growth hormone (GH) secretion from pituitary gland inhibiting the animal growth during stress response [18]. Stress can also have a harmful effect on the quality of food products of farm animals (meat, milk and eggs). Growth performance and product quality are com-promised in cows and small ruminants (sheep and goats) and also in pigs due to different stressors such as space allocation and heat stress [19-22]. In addition, cold stress in swine has been recognized as a cause of neonatal morbidity and mortal-ity [23]. There are experimental evidences that show how environmental stress in breeding can cause problems in ani-mal fertility [24]. In particular heat is a stressor that can strongly influence fertility of herds [25] and also influence meat quality. Christensen and colleagues demonstrated how milk toughness could be related to the different exposure of animals to non comfortable temperatures [26]. Both stressors produce a negative effect on dairy production and a consid-erable economic loss.

2.2. Impact of Stress and Environmental Adverse Condi-

tions on Susceptibility to Diseases

Stress conditions have been reported to suppress the im-mune system and may lead to an increased occurrence of disease in the presence of pathogens. The immune response is one of the mechanisms by which animals defend against different stressors or adverse environmental conditions. Acute and chronic stress tends to affect the immune re-sponses in different ways. Chronic stress often suppresses the immune system thus enhancing the susceptibility of an animal to disease. On the other side, acute stress often has no

158 Current Protein and Peptide Science, 2014, Vol. 15, No. 2 Bassols et al.

effect on immunity or even sometimes the immune system is enhanced by stress [27]. Stressors activate the hypothalamic-pituitary-adrenocortical (HPA) axis and the sympatho-adrenal medullary axis. Adrenocorticotropic hormone (ACTH) is the major hormone which regulates the synthesis and secretion of adrenal glucocorticoids. In turn, the secre-tion of ACTH in farm animals particularly cows, sheep and pigs is regulated by corticotrophin-releasing hormone (CRH) and vasopressin [28]. Additionally, some cytokines like in-terleukins and tumor necrosis factor have been found to be involved in pituitary ACTH secretion [29]. The most impor-tant mediators of the stress response are catecholamines, epinephrine and norepinephrine, which are the fast-acting hormones released by the sympathetic nervous system, and the slow-acting glucocorticoids cortisol and corticosterone, which are secreted by the adrenal gland after activation of the HPA axis [18]. The secretion of glucocorticoids and catecholamines in response to stress has a negative impact on functions of cells of the immune system in cattle and pigs and causes reduced lymphocyte proliferation, natural killer cells activity, neutrophil function and impaired interleukin and antibody production [30, 31]. Furthermore, glucocorti-coids upregulate the production of the anti-inflammatory cytokines IL-4 and IL-10 [32]. Also, the exposure of farm animals to different stressors, such as long distance transport and changes in nutrition is associated with an increase in serum acute phase protein concentrations [33-35]. All those changes make farm animals susceptible to pathogens with higher incidence of diseases. Particularly, stress of parturi-tion and lactation and also long-distance transportation in dairy cows is associated with increased susceptibility to in-fectious disease such as mastitis, paratuberculosis, salmonel-losis and bovine respiratory disease complex and other pro-duction diseases including infertility, uterine diseases (metri-tis, retained placenta, prolapsed uterus), ketosis and milk fever especially during periparturient period [36, 37]. Chronic stress in small ruminants, particularly sheep and goats, may result in poor welfare and impaired growth, milk production and reproduction and contribute to the develop-ment of diseases such as parasitism and enterotoxaemia [38, 39]. In pigs, stress of housing conditions and transportation could make pigs more susceptible to infectious diseases of gastrointestinal tract such as post-weaning colibacillosis, salmonellosis, swine dysentery and porcine proliferative enteropathies [40]. Also, modern pork production worldwide cope with porcine respiratory disease complex caused by multiple viral and bacterial pathogens including porcine re-productive and respiratory syndrome virus, swine influenza virus, porcine respiratory coronavirus, Mycoplasma hy-opneumoniae, Bordetella bronchiseptica, Pasteurella multo-cida, Actinobacillus pleuropneumoniae and others [41]. A number of stressors in pigs like marketing, vaccination, cas-tration, oestrus, parturition and climate conditions can trigger porcine stress syndrome, which is an inherited neuromuscu-lar disorder characterized by an abnormal accumulation of lactic acid in the muscle cells, with a high impact on mortal-ity as well as on meat quality and composition [42]. In addi-tion to suppression of the immune system, stress may also contribute to infections or inflammatory disorders of the gas-trointestinal system by physiological alterations in the gas-trointestinal tract. The enteric nervous system normally se-cretes norepinephrine, the major neurotransmitter in the gas-

trointestinal tract into the mucosa where a variety of bacteria are presented including food-borne pathogens [43]. Signifi-cant increases in stress mediators, particularly glucocorti-coids and catecholamines (epinephrine and norepinephrine), can affect the status of resident microbiota and disrupt the intestinal barrier function and increase mucosal permeability with a consequence of an increased microbial translocation rate in the gastrointestinal tract. Additionally, stress-related hormones can alter interactions between the mucosa and luminal bacteria affecting the commensal microbiota and leading to an increased passage of pathogenic bacteria [44]. It has been demonstrated that exposure to norepinephrine in vitro result in a significant increase in Gram-negative bacte-rial growth. In addition, stress-released norepinephrine in vitro enhances the virulence of enteropathogens such as Campylobacter and E. coli (reviewed in [45]). Consequently, stressed animals are more susceptible to bacterial infections and a number of farm animals (pigs, cattle and poultry) bring foodborne pathogens into the abattoirs. Moreover, the expo-sure to various stressors can increase fecal shedding of en-teric pathogens, such as E. coli, Salmonella and Campy-lobacter, leading to an increased cross-contamination during transport and to a higher degree of carcass contamination that is the major cause of foodborne disease with a high im-pact on human health [46]. These aspects will be analyzed in details in the chapter dedicated to foodborne pathogens.

2.3. Stress-related Oxidative Damage

Many stress conditions have been associated with oxida-tive stress like weaning, transport [47, 48], and diet [14, 15].

Chronic exposure to stress results in metabolic changes with damaging effects, in which production of reactive oxygen species (ROS) and oxidative stress play a major role. Stress hormones, norepinephrine and glucocorticoids, may contrib-

ute to enhanced formation of reactive oxygen species and oxidative stress [49, 50]. Oxidative stress is recognized as a process implicated in many pathophysiological conditions of farm animals. Aerobic respiration and metabolism generate

reactive oxygen species (ROS) as their by-products arising mostly from mitochondrial electron transport chain [51]. Cytochrome P-450 enzymes are an important source of ROS metabolizing either exogenous (xenobiotic) or endogenous

(physiological) substances [52]. ROS comprise two catego-ries of molecule species: free oxygen radicals and non-radical ROS. Free oxygen radicals are chemical species con-taining one or two unpaired electrons. Non-radicals do not

contain unpaired electrons but they are very unstable and can react with free radicals resulting in a new radical formation leading to chain reaction of free radical generation. Common free oxygen radicals include hydroxyl radical ( OH), super-

oxide anion ( O2-) and nitric oxide ( NO) while non-radical

include hydrogen peroxide (H2O2) [53]. ROS are not always harmful; they are physiologically used by cells in intracellu-lar signaling involved in gene expression, cell growth, pro-

tein synthesis, activation of receptors and others. However, an imbalance between ROS production and their safe re-moval by antioxidants leads to oxidative stress. Exceeded amount of ROS can modify cell functions and endanger cell

survival. ROS become stable by acquiring electrons from lipids, proteins, nucleic acids, and carbohydrates or any other nearby molecule leading to chain reactions of free radical


molecules formation. Oxidative damages of macromolecules

modify metabolic pathways and change the integrity and fluidity of cellular membrane resulting in oxidative cell in-jury, cell death and disease [54]. Among the biological mac-romolecules being subjects to ROS action, proteins are one

of the most important targets for oxidation and both the pro-tein expression and their modifications are affected by oxida-tive stress. Proteins undergo various damages; from protein cleavage by hydroxyl radical and protein crosslinking to

many side chain modifications. One of the most common modifications on side chains is carbonyl formation by the action of very reactive hydroxyl radical or less active oxi-dants, such as hydroxyl peroxide [55]. These oxidative dam-

ages lead to protein degradation and changes in protein struc-ture and function modulating important biological processes within the cells.

Different stressors, external or internal can contribute to ROS generation. Oxidative stress is considered as a notable component in the signaling processes involved in inflamma-tory responses including stimulation of cell adhesion mole-cules and production of chemo-attractant. During inflamma-tory conditions, phagocytes produce reactive oxygen species (ROS) that are needed for killing bacteria [56]. However, raised amount of ROS can overcome the antioxidant system and compromise the immune function of farm animals. Low level of antioxidants can diminish functions of lymphocytes and macrophages, i.e. their phagocytic ability and microbial toxicity [57]. In farm animals, oxidative stress is considered to be involved in pathological conditions relevant to animal production and general welfare of animals such as mastitis and reproductive disorders in ruminants, respiratory and en-teric diseases in cattle and pigs, parasitic diseases in small ruminants [58, 59]. Some studies have indicated that sup-plementation of dairy cows with antioxidants, selenium and vitamin E, enhanced macrophage function and production of macrophage-derived IL-1, thus improving the immune status of cows [58, 60]. It has been also suggested that selenium supplementation in sheep may reduce the susceptibility to parasitic invasion [61]. Oxidative stress has been found to be increased in transportation stressed cattle with a higher inci-dence in respiratory disease [62]; in cows stressed by parturi-tion and early lactation [63] as well as in cows with mastitis [59]. Oxidative stress in pigs increases the susceptibility to diseases, in particular respiratory infections [64]. It has been also found that even stress due to high density housing con-dition is related to oxidative damage in pigs [65].

3. PROTEOMICS TOOLS TO EVALUATE STRESS

CONDITIONS AND THEIR IMPACT ON ANIMAL

HEALTH AND WELFARE

The proteome analysis is in general a much greater chal-lenge than the transcriptome due to the different forms of proteins in different tissues and biological fluids, including post-translational modifications. Proteomic techniques have become an important tool for the study of molecular mecha-nisms in physiology and etiopathogenesis of diseases. As in human medicine, a great variety of proteomic approaches exists also in veterinary sciences, which can be grouped into two broad categories: gel-based assays and shotgun pro-teomics. The general advice, also in animal science, is to use different techniques to better understand the biological ques-

tion, as they are complementary. The ‘best technique’ for every sample does not exist.

3.1. Gel-based Approaches

Among the gel based approaches for the separation of proteins, the two dimensional electrophoresis (2-DE) re-mains at the moment one of the most used techniques due to its relative simplicity and an excellent resolving power [66]. In this technique proteins are separated first by isoelectric point and a further separation is performed by molecular weight. After the second step, proteins are detected using visible stains (colloidal Coomassie, silver stain) or fluores-cent (Sypro, Flamingo, Deep Purple). At this moment several quantitative limitations remain, due to the sample nature and low gel to gel reproducibility [67]. In the difference gel elec-trophoresis (2D-DIGE) the reproducibility problem has been overcame adding a labelling step prior to 2-DE with three amino-selective fluorescent dyes with different excitation wavelenghts (Cy2, Cy3 and Cy5). The use of an internal standard (Cy2 – labelled sample) eliminates gel-to-gel varia-tion and the parallel separation of different labelled sample (Cy3-Cy5) into the same gel provides excellent results with a high statistical power and a lower number of replicates [68]. To investigate quantitative differences between samples, stained or fluorescent gels have to be digitalized and the im-ages analyzed with a dedicated software (Progenesis, Decy-der, Delta 2D). The software workflow integrates sophisti-cated statistical tools to perform uni- and multi-variate analysis to simplify complex data elaboration. Identification of protein spot usually is done by tryptic digestion of pro-teins and peptide analysis by PMF (peptide mass fingerprint-ing) using MALDI-TOF (matrix assisted laser desorption ionization- time of flight) mass spectrometry (MS). Using PMF protein identification is based only on the mass match-ing of peptide. To obtain a unique structural information about the peptides analyzed it is necessary to fragment ion-ized peptides into the mass spectrometer in MS/MS mode to achieve the full sequence and to infer with high confidence the corresponding protein.

3.2. Gel-free Approaches

During recent years gel-free approaches have become very popular due to the advent of more complete databases and accurate spectrometers with the advantage also of the high throughput analysis. The quantitative analysis of pro-teomes by MS is a crucial point of gel-free approaches and currently two different methods are used, label-free and la-bel-based [69]. In the first one unlabeled proteins from a sample are digested into peptides and separated prior to iden-tification by MS. The acrylamide gel as a separating system has been replaced by a liquid chromatography (LC) step at high or ultra- high pressure (HPLC or UHPLC) with normal or nano volumes. The most frequently used LC-method nowadays is the reverse phase chromatography where pep-tides are separated according to their hydrophobicity on non-polar columns (C18). To augment resolution it is possible to couple a second dimension of separation (2D-LC) usually using a polar column (ion-exchange) that separate peptides according to charge. After the acquisition of peptide spectral signals on different mass analyzers (TOF, Orbitrap, FTMS) the relative amount of all proteins in two or more samples of


interest is calculated using the corresponding peptide inten-sity between the LC-MS runs [70]. On the other hand the label-based approach use different pairs (heavy and light) of isotopic mass-tags covalently linked to the proteins that can be easily detected by mass spectrometry. Depending on the type of labelling this strategy can be divided into two more classes: metabolic labelling and chemical labelling [71]. The metabolic labelling is an in-vivo labelling technique where cells or organisms are labelled with different isotopic mole-cules (salts and/or amino acids) during growth.

14N/

15N la-

belling refers to the use of inorganic ions to label cells and the MS quantitation is done between the ratio of labelled (

15N, heavy) and unlabelled (

14N, light) peptides obtained,

for example, from different experimental classes (con-trol/diseased). In SILAC labelling (stable isotopic labelling with amino acids in cell culture) a pair of isotopic aminoac-ids, usually light and heavy lysine or arginine, are used to label the cells. In the chemical labelling strategy proteins or peptides are isotopically or isobarically tagged using a chemical reaction [72]. Due to the multitude of chemical features (label and reaction site) that can be used for the la-belling, several strategies have been developed. Among the overall amount of approaches iTRAQ and ICAT are the most used. In the iTRAQ (Isobaric Tags for Relative and Absolute Quantification) approach is possible to compare up to eight samples in the same experiment. The tags used are isobaric and the quantification is done using reporter ions intensities obtained in the low mass range (m/z 114-121) after the fragmentation of the differentially labelled peptides in MS/MS mode [73]. The ICAT (Isotope-Coded Affinity Tags) approach combines a selective cysteine-reactive group with light and heavy isotopic tags (8-Da shift) and a biotin-tag. The latter one is used to purify labelled peptides before the MS quantitation based on ion intensities of isotopic tags [70].

3.3. Role of Serum Proteomics in Animal Health Studies

Several proteomic studies highlighted useful results for diagnosis, pathogenesis, prevention and treatment of com-mon veterinary diseases. This type of results will be de-scribed in the following section.

3.3.1. Pigs

From an economical point of view, the most harmful dis-eases in pig production are PRRS (porcine respiratory and reproductive syndrome) and PMWS (post-weaning multisys-temic wasting syndrome).

The etiological agent of PRRS is the PRRS virus (PRRSV), a virus that belongs to the genus Arterivirus in the family Arteriviridae and the order Nidovirales. PRRS is clinically characterized by reproductive losses in late gesta-tional sows, increased number of weak or stillborn pigs, se-vere pneumonia in neonatal and nursery pigs, reduction in growth performances and increased mortality at all ages. The innate immune response against PRRSV is relatively weak since one of the defence mechanisms of the virus is to ac-tively suppress the immune responses and the production of several key immune-regulatory cytokines [74-76]. Several proteomics studies have provided information about changes induced by the virus infection in macrophages, which are the cells where the virus preferentially infects and replicates [77]

or lungs [78]. In macrophages two proteins involved in stress response, heat shock 27 kDa protein (HSP27) and superoxide dismutase 2 (SOD2) were up-regulated [77]. Post-weaning, multi-systemic wasting syndrome (PMWS) is presently a major disease problem with significant welfare and eco-nomic consequences for pig producers. The disease is char-acterized by wasting, respiratory, enteric and lymphoid sys-tem problems in young pigs of 4-16 weeks of age [79]. PMWS is considered to be caused by infection by the PCV2 virus, although other factors could be associated to the trig-gering of the disease. Proteomic studies have shown that infection of macrophages by the virus causes changes in pro-teins related to stress response revealing the implication of oxidative stress in the pathology [80].

Other viral diseases of high interest in porcine production are Classical swine fever (CSF) and African swine fever (ASF). CSFV belongs to the genus Pestivirus within the family Flaviviridae. Virulent strains of classical swine fever virus (CSFV) provoke severe disease in pigs characterized by immunosuppression, thrombocytopenia and disseminated intravascular coagulation, which causes significant economic losses to the pig industry worldwide [81, 82]. The proteomic analysis by 2D-DIGE of serum from experimentally infected pigs at a stage with almost no clinical signs revealed differ-ential expression of up-regulated and down-regulated pro-teins in CSFV-infected pigs, involved in blood coagulation, anti-inflammatory activity and angiogenesis. These proteins with altered expression may have important implications in the pathogenesis of classical swine fever [82]. Proteomic technology was also used to globally examine ASFV-infected cultured Vero cells in order to determine target pro-teins for the virus. After 2-DE, the most significant changes were in redox-related proteins, heat shock proteins and apol-ipoproteins. These cellular protein modifications could rep-resent distinct roles during infection related to apoptosis and transcriptional modulation mechanisms [81]. In both cases, they may provide a clue for identification of biomarkers for classical and African swine fever early diagnosis. Foot and mouth disease (FMD) is another important disease in farm animal production. Proteomics have been used to analyze serum protein differences between normal and FMD virus-infected piglets by LC-MS/MS. Comparing the serum pro-tein composition before and after FMDV infection, several proteins were found to be expressed after FMDV infection in the same piglet (apolipoprotein A-IV precursor, haptoglobin and probable chemoreceptor glutamine deamidase cheD) [76]. Authors proposed that these results may provide further information about biomarkers for early diagnosis of FMD in piglets. The immune response against a pathogen could be also explored in the search of immunoreactive proteins, which could be potential candidates for vaccine production. This strategy consists usually in the separation of the pro-teins from a pathogen extract by a 2-DE followed by a west-ern blot with the serum of diseased or challenged animals [83]. This technology has been used to identify immunoreac-tive proteins against Haemophilus parasuis (Glässer’s dis-ease) that are considered novel immunogens and candidates in future vaccines [84]. This approach has also been used to identify proteins differentially expressed by pathogenic and non-pathogenic strains of Streptococcus suis. S. suis is an important swine pathogen with worldwide distribution that is also responsible for a variety of human diseases. Two studies


have reported the identification of several proteins expressed only by pathogenic strains and thus they may be considered putative virulence-associated factors [85, 86]. Diarrhea is a major challenge in industrial production of pigs, particularly in neonatal and weaning piglets, being Salmonella typhi-murium a major cause. The immune reaction of the pig against the pathogen has been studied in mesenteric lymph nodes (MLN) of pigs after S. typhimurium infection by DIGE-based proteomics. The proteome response of porcine MLN to infection was associated to the induction of proc-esses such as phagocyte infiltration, cytoskeleton remodeling and antigen presentation. These results may help to under-stand the host innate and adaptive immune response of the animal to control S. typhimurium dissemination in swine infections [87].

To prevent diarrhea with nutritional supplementation is a

usual procedure in porcine farms. Zinc oxide (ZnO) is an important dietary factor that regulates intestinal amino acid and protein metabolism in animals and, given as a supple-ment to the diet, ameliorates weaning-associated intestinal

injury and growth retardation. Proteomics (2-DE and MS) helped to understand the underlying mechanisms since it was shown that zinc supplementation affects the expression of proteins related to glutathione metabolism and oxidative

stress in the gut. Consistent with the changes in protein ex-pression, the ratio of reduced glutathione to oxidized glu-tathione was increased, whereas glutathione-S-transferase and glutathione peroxidase activities were reduced in ZnO-

supplemented piglets, indicating that ZnO supplementation improves the redox state and prevents apoptosis in the jeju-num of weaning piglets, thereby alleviating weaning-associated diarrhea [88]. In neonatal mammals, the lack of

colostrum is also a frequent cause of diarrhea, since the up-take of intact immunoglobulins (IgG) from colostrum is critical for the acquisition of passive immunity and the pre-vention of intestinal and systemic diseases. The mechanisms

of uptake, the isoforms of IgG taken up by the healthy tissue and the failure of inflamed gut tissues from pre-term piglets were also investigated by 2-DE and MS [89].

3.3.2. Cattle

In cattle, one of the major health problems is represented by mastitis, which is an infection caused by a variety of pathogens invading the lactating mammary gland, whereas brucellosis, tuberculosis and viral diseases are most relevant for their effects on meat production [6]. Several proteomics studies on serum have been performed in order to detect pu-tative biomarkers useful for the subclincal diagnoses of mas-titis [59, 90]. Mastitis is caused by a variety of pathogens invading the mammary gland. The annual cost associated with treatment, culling and death of cows as well as loss in milk production due to mastitis is estimated to be £168 mil-lion in the UK alone [91]. Furthermore, mastitis severely compromises the welfare of cows. Early diagnosis and iden-tification of the causal pathogen are crucial for initiating a successful antibiotic treatment, ensuring fast recovery and thereby minimizing the impact on animal health [6]. The serum proteome of cows affected by subclinical and clinical mastitis has been addressed above, and a systemic inflamma-tory and oxidative stress response has been observed in af-fected animals, being acute phase proteins an important

component of the early phases of the disease [59, 90]. Bru-cella is a Gram-negative, facultative intracellular bacterium that is the etiologic agent of bovine brucellosis and causes chronic infection in humans. In livestock the disease is char-acterized by abortion and sterility. Proteomics helped to characterize the immunome of Brucella strains to explain host specificity and virulence differences among Brucella species. Thus, the host response to infections with virulent or attenuated Brucella strains has been analyzed in macro-phages and other cell types and many virulence factors have been identified [92]. Proteomic techniques, generally based in two-dimensional electrophoresis, have enabled the charac-terization of proteins that could be used as tools to develop sensitive and specific immunoassays for serodiagnosis of bovine brucellosis [93], and to identify candidate proteins for developing better vaccines against Brucella infection in bo-vine and humans [94]. Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), characterized by se-vere emaciation that poses a significant economic problem to the beef and dairy industry worldwide. Although there are several techniques available for diagnosis of JD, their sensi-tivity is questionable. A combination of several techniques (2D-DIGE, LC-MS/MS, iTRAQ) has been useful for novel biomarkers of MAP infection [95], to characterize the differ-ential proteome of Type I and Type II MAP, which are dis-tinct and appear to have different host preferences [96], and to provide further information about the pathogenesis of the disease [97]. An immunoproteomic approach has also helped to identify candidate proteins for use antigens for improved serodiagnostic tests for bovine paratuberculosis [98, 99]. The background infection with MAP is high and it may confound the detection of bovine tuberculosis caused by similar myco-bacteria (M. bovis). In this case, a proteomic approach has been used to detect differentially expressed proteins in the serum of animals infected by one or the other pathogen, con-tributing to the development of mycobacterium specific tools [100]. Viral diseases are also important pathologies in cattle. Bovine viral diarrhea virus (BVDV) infection is widespread in cattle worldwide, causing important economic losses. Pathogenesis of the disease caused by BVDV is complex, due to the existence of several strains and biotypes. BVDV can cause a persistent latent infection and immune suppres-sion if animals are infected during early gestation. The mo-lecular mechanisms that underscore the complex disease etiology leading to immune suppression in cattle caused by BVDV have been addressed by evaluating the effect of BVDV infection of bovine monocytes to determine their role in viral immune suppression and uncontrolled inflammation [101, 102]. Other cattle diseases that have been studied by using proteomics are claw horn disruption (CHD), a common underlying cause of lameness in dairy cattle which leads to compromised animal welfare and production losses, with the characterization of three different claw tissues [103] and milk fever, an important metabolic disorder of dairy cows after calving, characterized by hypocalcemia, tetany, lateral recumbency, and eventual coma [104].

3.4. Role of Serum Proteomics in Animal Welfare Studies

Proteomics has been used to identify new potential stress biomarkers in pigs housed at different densities since insuffi-


cient space allowances induce a repeated state of stress that alters the activity of the pituitary–adrenal axis, behavior and reproduction [105, 106]. Elsewhere, it was shown that the use of 2D-DIGE allowed the identification of actin as a po-tential biomarker of stress [65]. 2D-DIGE was also used to analyze the serum proteome of cows maintained under dif-ferent management systems (good and semiferal conditions), showing an involvement of the redox system as the main adaptation of cows living in challenging environmental con-ditions [107].

Likewise, the stresses of transportation, weaning and

commingling are associated with an increased incidence of

bacterial and viral pneumonia in cattle. The interaction be-tween both conditions has been examined by 2-DE in the

epithelial lining fluid of bovine respiratory tract, showing

that stress causes protein changes in pulmonary fluids, sug-gesting a mechanism through which stress alters respiratory

disease susceptibility [108]. Similarly, the proteomes of bo-

vine serum samples have been characterized and used to-gether with other OMICS to discriminate between the bio-

logical responses to stress or infection (bovine respiratory

disease (BRD)), showing that the changes produced in the serum profile are different depending on the causing effect

[62].

Nutrition is an important component of welfare. In dairy cows, ketosis is a common metabolic disorder that is fre-quently observed during the early lactation period. Evalua-tion of oxidative stress has increasingly contributed to the

understanding of the fundamental mechanisms involved in metabolic disorders [57, 63]. The molecular pathways acti-vated in the liver of the ketotic cow were also studied by 2-DE coupled to MALDI-MS/MS to compare the liver pro-

teomic profile between ketotic and normal cows. The differ-entially expressed proteins play a role in energy metabolism, carbohydrate degradation, fatty acid metabolism, amino acid metabolism, antioxidation, cell structure, nucleotide metabo-

lism, and protein metabolism [109]. Ketosis is due to a nega-tive energy balance of the individual, and appears when the energy demand for maintenance and lactation exceeds that of dietary energy intake, very frequently around calving. The

hypothalamus is the central regulatory unit that balances a number of body functions including metabolic rate, hunger, and satiety signals, and respond to alterations of circulating nutrients and hormones that reflect the peripheral energy

status. To understand such a control, the hypothalami from fed and energy restricted cows were characterized using pro-teomic techniques, leading to the identification of several proteins involved in energy and nucleotide metabolism, and

oxidative stress under conditions of dietary energy defi-ciency [110, 111]. Other researchers have focused their at-tention on the effects of body weight and nutrition on mam-mary parenchymal tissue protein expression profiles in heif-

ers’ mammary development by using a proteomic approach [112].

In pigs, the relation between the type of diet and the oxi-dative status of the individual has been also studied by pro-teomics. Thus, a diet based on a soy protein diet (SPI) sig-nificantly changes the hepatic transcription pattern compared with a casein diet showing an increased oxidative stress re-sponse and significant effects on protein biosynthesis [113].

The possibility that maternal diets during gestation could affect growth and tissue development of offspring and pro-gram their later phenotype is an emerging challenge in pig production. This issue has been addressed by investigating the effects of protein levels in diets of pregnant sows on the proteomic features of subcutaneous adipose tissue (SCAT) of the offspring at birth and its possible persistence later in age (6 months old). Modifications in SCAT protein abundance shortly after birth were investigated by 2-DE and MS. In this study, a higher abundance of proteins involved in pathways related to glucose and fatty acid metabolisms, lipid transport, and regulation of apoptosis were found in low protein-piglets in comparison with control piglets [114]. Finally, the mo-lecular expression changes that occur during pregnancy were analyzed in porcine peripheral blood mononuclear cells (PBMC) using proteomic analysis. By classifying the pro-teins according to their functions, a large number of differen-tially regulated proteins involved in anti-oxidant, detoxifica-tion and stress response pathways were found, upregulated during pregnancy [115].

3.5. Proteomics of Other Body Fluids in Farm Animals

Serum and plasma are the most frequently used biologi-cal samples for monitoring health and disease in farm ani-mals. Blood is an easy sample to obtain, but increasing atten-tion is being paid Animal welfare to other, noninvasive sam-ples, such as milk and saliva, since their compositions may reflect the overall health status of the individual animal [116-118].

3.6. Milk

Comparative proteomic analyses of bovine milk have emerged in recent years [8]. Milk and whey have been thor-oughly studied in mastitic cows. Normal and mastitis whey showed a very different composition, likely due to extravasa-tion of blood proteins to the mammary gland [119]. Different isoforms from the most abundant protein in milk, casein, were detected in both normal and mastitis whey. Other pro-teins, such as lactotransferrin, serotransferrin and many cel-lular proteins were only detected in the inflamed animal samples. They are responsible for the great change in com-position between normal and mastitis whey, especially those which exert a biological function related to immune defense [90, 120]. The differential response to infection by Gram-negative and Gram-positive bacteria has been analyzed in whey from cows naturally infected with E. coli or S. aureus [121, 122]. S. aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical but can lead to chronic infection, due to the pathogen's ability to evade the innate defense mechanisms. Proteomic analysis of the milk following intramammary infection revealed unique host pro-tein expression profiles that were dependent on the infecting strain as well as the phase of infection, and allowed the iden-tification of potential antimicrobial peptide involved in host defense [123]. Intramammary infusion of lipopolysaccharide (LPS) that simulates mastitis caused by Gram-negative bac-teria and mammary epithelial cells stimulated with heat inac-tivated E. coli o S. aureus strains have been also used as ex-perimental approaches for the study of the pathogenesis and biological response in mastitis [59, 121, 124].


3.7. Saliva

The benefit of saliva as a specimen is that sample collec-tion is not invasive, hence allowing frequent sampling. Sa-liva is an extraordinary fluid in terms of research and diag-nostic possibilities. Its composition in electrolytes, hormones and especially its proteome contains information about feed-ing status, nutritional requirements and adaptations to diet and environment, and also about health status of animals. Therefore, the analysis of salivary proteomes is emerging as a tool to identify animal disease biomarkers and to better understand animal physiology [116-118, 125].

4. FOODBORNE PATHOGENS AND PROTEOMICS

Food safety encloses different aspects, from genetically

modified organisms (GMO), to illegal treatment of animals up to foodborne pathogens that can produce toxins danger-

ous to humans. Proteomics applied to the study of food can

help to counteract such public health problems, in particular those related to the presence and growth of foodborne patho-

gens. Fig. 3 resumes the current applications of proteomics

in the field of microbial food safety; most of literature de-scribes the immunoproteomic approach to detect specific

immunoreactive epitopes that can be detected through the

application of a targeted (SRM- MS MS) analysis of proteo-typic peptides of bacteria of interest.

4.1. Salmonella spp.

Salmonella spp. colonise all the major livestock species and very often food-producing animals, which may be as-ymptomatic. This represents a problem because of the possi-ble production of contaminated meat and other food prod-ucts. Furthermore, Salmonella is an adaptable microorgan-ism that could be present in fresh products and also survive

in presence of preservatives and additives. Calhoun and col-leages [2] described, using a comparative proteomic ap-proach, the effect of propionic acid (PA), a common food preservative agent, on the metabolism of Salmonella. This work provides a comprehensive analysis of differentially expressed proteins between the unadapted and PA adapted condition. Furthermore, the study of variation of protein ex-pression on PA adapted cells, gave new insights to describe new putative virulence factors of this organism.

Another interesting paper of Kim and colleagues [126] describes the setup of a quantitative method to detect Salmo-nella spp. with a modified SILAC procedure. Authors identi-fied and reported 76 proteins from a strain of Salmonella enterica serovar Enteritidis, which were differentially ex-pressed during exposition to H2O2 oxidative stress. The final result is a protein, SP1, that could be very interesting as marker of systemic infection in an animal host.

4.2. Campylobacter spp.

On the basis of data available from European Union (EFSA), Campylobacter spp. is considered the major cause of acute food poisoning mediated by bacteria [127]. Fur-thermore, the incidence of this foodborne pathogen varies between years and countries for unknown reasons. Poultry is the major reservoir of this pathogen, and C. jejeuni, the more representative of this class, cannot grow outside a host. Fur-thermore, Campylobacter has a strong capacity to colonise new niches because of its formidable mechanisms of adapta-tion to different environmental stressors, including antibiot-ics. Due to the unclear mechanism of infection, the majority of literature about Campylobacter is focused on the elucida-tion of infection mechanisms, as reported, for example, by Elmi and colleagues. [128]. These authors used proteomic techniques to identify outer membrane vesicles proteins

Fig. (3). General strategies of proteomics approaches useful for foodborne pathogen investigation.


(OMV) and investigated the mechanisms of interaction be-tween Campylobacter OMV and intestinal epithelial cells, with particular attention to the induction of the host innate immune response. Authors discovered the presence of N-linked glycoproteins associated with OMV participating in this mechanism. Liu and colleagues [129] performed a pro-teomic analysis of proteins extracted from different C. jeje-uni isolates by LC-MS/MS and demonstrated a remodelling of the C. jejuni proteome that occurs within cultured mam-malian cells, attributable to specific adaptation to the intra-cellular environment. This fact may explain the difficulties to growth this pathogen under standard laboratory conditions.

4.3. Listeria

Listeria monocytogenes is an opportunistic foodborne pathogen that can cause listeriosis. It has been estimated that in the United States alone is responsible for 28% of all food-related deaths [130]. The most susceptible targets are im-munocompromised patients, neonates and pregnant women. The more frequent reservoirs of L. monocytogenes appear to be soil and mammalian GI tracts that contaminate vegetation. Grazing animals can ingest the microorganism and further contaminate vegetation and soil. This is a disease character-ized by oral-fecal transmission. One of the most dangerous features of these bacteria is its adaptative capacity. A pro-teomic study demonstrated how this microorganism is ex-tremely versatile in its adaptive mechanisms to cold. The study highlighted that protein synthesis and folding, nutrient uptake and oxidative stress pathways were the most impor-tant pathways involved in cold adaptation response. The gained knowledge is important to evaluate the possibility of an intervention to counteract its growth at cold temperatures [131]. Moreover, the differential protein expression of L. monocytogenes pathogen growing under the presence of high concentrations of bile salts has been described, showing dif-ferences in the expression of proteins involved in biofilm formation, transmembrane efflux pumps and osmotic stress response. This data demonstrates how variability among strains could be a key component of Listeria virulence [132].

4.4. E. coli

At least six groups of E. coli have been isolated from the gut of mammalian hosts (humans and other animals); among them Vero cytotoxin producing E. coli (VTEC strains) and E. coli O157:H7, which are widely recognized as important cause of foodborne disease in the last two decades. Cattle are the most important reservoirs of E. coli O157:H7 that is the major cause of outbreaks in humans. It has documented how most outbreaks are indeed due to improper food handling practices and to the consumption of undercooked meat [133, 134]. These strains of E. coli are normally present in the gut of asymptomatic cattle but they can often contaminate beef meat, minced and other derived products for human con-sumption, including milk [134].

As other foodborne pathogens, E. coli has the ability to continuously evolve into new types that are never character-ized. From this point of view, proteomics is an important tool to understand the mechanisms of evolution of this pathogen. The proteome of E. coli has already been well characterized [135]. An important problem related to this infection is antibiotic resistance, a phenomenon that has been

associated to the treatment of animals with sub therapeutic doses of antibiotics as a preventive measure in farms. Pro-teomics, in particular 2D-DIGE coupled with MS, have been used to study the effect of triclosan, a disinfectant used for several strains of wild type and E. coli O157:H19. Interest-ingly, authors described that triclosan tolerance acts trough several concerted mechanisms to achieve high-level resis-tance, involving quorum sensing pathways [136].

Another interesting work that attempts the elucidation of the of antibiotic multiresistance mechanism, a big issue in

animal breeding, has been reported by Piras and colleagues

[137], who used a proteomic approach based on 2-DE cou-pled with MS/MS to reveal that antibiotic multiresistance

(more than 4 antibiotics) in E. coli from buffalo species in-

volves proteins of quorum sensing, such as autoinducer II and Lux S.

CONCLUDING REMARKS

It is clear that numerous aspects can contribute to shape the entire chain from farm to fork. About this topic animal

stress and animal welfare are strictly linked to the subsequent

food safety and quality. Proteomics represents a relatively new ‘way of thinking’ that, thanks to the dramatic increase

of innovative techniques, is able to help in the discovery of

new ways to improve general management of animals, wel-fare, monitoring immunoresponse and faster diagnosis of

disease with particular attention to subclinical diseases. Last,

but not the least, microbial proteomics is a valid method to revolutionize the classic approach to diagnosis. Through

proteomics it is possible to understand the mechanism of

infection and consequently to counteract it, earn money and avoid the use of antibiotics (and mutiresistance). Proteomics

should be considered a valid help for public health, and can

be adopted to upgrade and improve some protocols that could be used in official surveillance and test development.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

The authors are grateful to COST ACTION FA1002- Farm Animal Proteomics- for network provided.

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Received: October 13, 2013 Revised: October 13, 2013 Accepted: November 19, 2013

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