**3. The metabolomics study of its applications**

the manifestation of clinical signs [19]. This association is a result of the greater intensity of parasite load, when there is a lower production of Th1 cytokines (Lymphocyte T helper type I) in various organs such as lymph nodes [20], spleen [21], bone marrow [22] and skin [23].

Animals that produce Th1-type response develops immunity against the parasite cell type, resulting in resistance to infection [19]. In these animals there is a predominance of IFN-γ, TNFα and IL-12 release, which among other things promotes the production and activation of

Resistant dogs are characterized by low parasitic burden in several organs [25], no develop‐ ment of clinical signs [19], normal erythropoietic and leukopoietic activities [22] and low

On other hand animals that produce Th2-type (Lymphocyte T helper type II) response are characterized by a humoral immune type response and consequent susceptibility to infection [19]. In these animals there is a predominance of IL4, IL10 and TGF-β that inhibit the production of IFN-γ and IL-2 then the cell type response [22, 27]. The antibodies produced by humoral response are unable to eradicate the infection resulting in high parasitic burden and expression

Thus in dogs with active VL the strength of parasite load is directly proportional to the intensity of clinical signs and disease severity (Figure 1). The most important clinical signs are ocular lesions, periocular and cutaneous, kidney diseases and their consequences; respiratory, digestive, cardiovascular and musculoskeletal disorders; hypertrophy of lymphoid organs and

The transmission of *Leishmania* by dogs seems to be related to the extent of clinical manifestations of disease so how much more clinical signs present higher the ratio of infectivity, which is possibly related with high parasite load in different organs, especial‐

Histological skin evaluations of animals which show the presence of perifollicular dermatitis with inflammatory infiltration generally extending by the sebaceous glands [30] and a predominance of macrophages, lymphocytes and polymorphonuclear neutrophils [31].

The hair is considered an appendix of the skin performing various functions in the body among which are included the secretion and excretion of endogenously produced substances by

The emission and accumulation of chemical compounds by hair are well-studied, in both humans and animals, for several purposes, either as indicative of the internal metabolism of drugs [33]; for investigation in toxicological forensic [34]; for evaluation of chemical contam‐ inants [35]; post-treatment accumulation of drugs [36] ; alcohol and drugs abuse [37]; evalua‐

Hair is an interesting sample for standardization of biomarkers because of its role to excrete endogenous substances [5]. Furthermore, the exam of dogs hair allowed to find *Leishmania*

DNA by standard PCR technique, reingorcing its importance for the diagnosis [40].

tion of trace elements [38]; or for anti-doping investigations [39].

macrophages, nitric oxide and cell apoptosis [24].

318 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

infectability to phlebotomine [26].

of clinical signs of disease.

hematological disorders [28].

ly skin and lymph nodes [29].

several metabolic processes [32].

Due to the current compartmentalization model widespread in human medicine and in veterinary gradually been developed a quest for a embracing perspective in life sciences. This trend is a throwback to ancient times where a holistic perspective was predominant [43]. Biological system, which brings together these ideas, is a term that has a recent origin and currently allows different types of research [44]. This attempt to an expanded approach began what the authors call the "omics" age, where genomics, proteomics and metabolomics studies has been carried out (Figure 2) [45].

**Figure 2.** The central dogma of biology and the omic cascade. Adapted of [45].

By definition, genomic characterizes hereditary information of an organism and defines what can happen in a biological system. On other hand, research about proteomics defining the structure and function of the proteins encoded by the genome of an organism is the next logical step for the study of biological systems. Finally, metabolomics systematically analyzes a whole set of chemicals resulting from specific cellular processes, determining the main aspects of the function and regulation of these processes [46].

Metabolomics is also called metabonomics and outlines the study of metabolome [47]. Metabo‐ lome in turn is a set of metabolites produced or present in a biological system [47]. Thus, metabolomics can be defined as the study that enables the detection, identification and quanti‐ fication of the largest number of possible metabolites in biological samples, searching for variations that can be used to discriminate volatile organic compounds between samples [48].

Metabolomics aims to determine a whole set of low molecular weight compounds (less than 1.500 Da) in a given biological sample, getting closer as much as possible of the physiology of the whole system [49]. For samples obtained from healthy subjects and patients, the aim is to identify one or more endogenous metabolites found in clinical samples related to disease [48].

This approach of combining various faces of a biological system allows integrating different details of this complex information network. Thus, this makes possible application of metab‐ olomics in different approaches, such as the study of basic biological models, drug develop‐ ment, human and animal pathophysiological models, toxicology studies, nutritional effects, metabolic consequences from genetic modifications, inborn errors of metabolism and identi‐ fication of biomarkers of diseases [45, 47, 50, 51].

Currently, analysis involving metabolomics has been successfully performed in several pathogens that infect living beings, such as lineages of uropathogenic *Escherichia coli* [52], Hepatitis B [53], the pathogenic fungi *Aspergillus fumigatus* [54] and protozoan *Plasmodium falciparum*, the major cause of malaria [55]. According Lakshmanan and Daily [50], metabolo‐ mics offers a unique and powerful tool to explore the biology of insects vectors and to develop strategies to stop its transmission. It has been shown that in the case of malaria some chemical compounds play a critical role in the development life cycle of *P. falsiparum* within *Anopheles* spp. vector and the interruption of the formation of such metabolites could help to reduce disease transmission [56].

what the authors call the "omics" age, where genomics, proteomics and metabolomics studies

By definition, genomic characterizes hereditary information of an organism and defines what can happen in a biological system. On other hand, research about proteomics defining the structure and function of the proteins encoded by the genome of an organism is the next logical step for the study of biological systems. Finally, metabolomics systematically analyzes a whole set of chemicals resulting from specific cellular processes, determining the main aspects of the

Metabolomics is also called metabonomics and outlines the study of metabolome [47]. Metabo‐ lome in turn is a set of metabolites produced or present in a biological system [47]. Thus, metabolomics can be defined as the study that enables the detection, identification and quanti‐ fication of the largest number of possible metabolites in biological samples, searching for variations that can be used to discriminate volatile organic compounds between samples [48].

Metabolomics aims to determine a whole set of low molecular weight compounds (less than 1.500 Da) in a given biological sample, getting closer as much as possible of the physiology of the whole system [49]. For samples obtained from healthy subjects and patients, the aim is to identify one or more endogenous metabolites found in clinical samples related to disease [48].

This approach of combining various faces of a biological system allows integrating different details of this complex information network. Thus, this makes possible application of metab‐ olomics in different approaches, such as the study of basic biological models, drug develop‐ ment, human and animal pathophysiological models, toxicology studies, nutritional effects, metabolic consequences from genetic modifications, inborn errors of metabolism and identi‐

Currently, analysis involving metabolomics has been successfully performed in several pathogens that infect living beings, such as lineages of uropathogenic *Escherichia coli* [52], Hepatitis B [53], the pathogenic fungi *Aspergillus fumigatus* [54] and protozoan *Plasmodium falciparum*, the major cause of malaria [55]. According Lakshmanan and Daily [50], metabolo‐ mics offers a unique and powerful tool to explore the biology of insects vectors and to develop

has been carried out (Figure 2) [45].

320 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

**Figure 2.** The central dogma of biology and the omic cascade. Adapted of [45].

function and regulation of these processes [46].

fication of biomarkers of diseases [45, 47, 50, 51].

In the case of *Leishmania* sp., most studies involving metabolomics deal mainly with metabolic changes in parasite at cellular level, differences in metabolism pathways of substances, sensitivity and resistance to drugs and other approaches related to the development of the parasite [57-59]. To our knowledge the only work that involves metabolomics with diagnostic purposes was developed in dogs with different clinical profiles for canine visceral leishma‐ niasis and it was found some of the volatile organic compounds emitted by animals such as potential biomarkers of disease [42].

Metabolomic studies in order to diagnose diseases are quite conducted for other diseases [2, 60]. The idea that chemical components resulting cellular activities can reflect the health status of an individual is old: in China (1500-2000 BC), doctors used ants to detect the presence of glucose in urine of patients with suspected diabetes [43]. The pathophysiological changes observed from metabolomic have a shorter response time in comparison to the development of clinical symptoms for example. This is one of the reasons why metabolomics has been used in an attempt to identify biomarkers for early stage disease, especially those of a chronic nature such as cancer and respiratory diseases, which usually have a later diagnosis [61, 62].

As in metabolomics studies takes into account a systemic approach, theoretically any change in the body, whether physiological or pathological, can influence the results. Thus, to minimize interference unrelated to the objectives of the study it is important that at the experimental design taken into account factors intrinsic and extrinsic to individuals, such as genetic variations, diet, gender, age of individuals, climatic conditions, concomitant infections, among others. Moreover, this multiplicity of effects can be used. For example, metabolomics can be used to identify the impact of nutrients in the diet on the biochemistry of living organisms and their susceptibility / resistance to disease [50].

The metabolites are a wide range of molecular arrangements and that provides a variation in several physical and chemical properties of their components. The degree of diversity is indicated by analysis of organic metabolites with low molecular weight, polar, volatile such as isoprene as nonanol, by analysis of metabolites with higher molecular weight, polar (carbohydrates) and nonpolar (terpenoids and lipid) [63]. To characterize and quantify these compounds is necessary to use specific methodologies and instruments according to the characteristics of each class. Therefore, metabolomics includes several analytical technologies that need to be carefully selected in accordance with the metabolites and metabolic pathway of interest, or the biological question to be answered.
