Cytokine concentrations and parasite copy number (×107)/ml in dogs with visceral leishmaniasis in different clinical staging.
\r\n\tfires. Infrastructure development and human settlements are blocking migratory corridors, thus suppressing their critical role of maintaining the ecological processes including allowing for the movement of animals and the continuation of viable populations. Human-wildlife conflicts are growing as a
\r\n\tresult of population growth and, therefore, increasing demand for natural resources and increased proximity to wildlife.
\r\n\tExotic and invasive species are outcompeting native species for resources or habitat and altering community structure with detrimental consequences on native species.
\r\n\tImpacts of climate change on wildlife are increasing and manifested through habitat destruction, increased human-wildlife conflicts, diseases and death of wild animals. Illegal and unsustainable hunting is increasing to cater for subsistence and commercial demands within and outside the national boundaries, thus leading to a dramatic drop of populations or extinction of species.
\r\n\tChapters presented in this book attempt to respond to four questions: How do the political, economic, social, ecological and technological changes influence the survival of wildlife? How do these changes shape the management policies and approaches? How effective are the existing strategies and options to coping with these changes? Which are the possible options to address these changes and secure a future for wildlife?
\r\n\r\n\tThis Book serves as an important platform for information and experience sharing among the scientists, academicians, students, conservationists and policy-makers from different parts of the world.
",isbn:"978-1-83880-976-8",printIsbn:"978-1-83880-975-1",pdfIsbn:"978-1-83880-977-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"a27827009edc70af81e12c10aa3e51dd",bookSignature:"Prof. Jafari Kideghesho",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8834.jpg",keywords:"Wildlife, Conservation, Poaching, Law Enforcement, Protected Areas, Wildlife Habitats, Migratory Corridors, Wildlife Diseases, Invasive Species, Wildlife Utilization, Human-Wildlife Interactions, Wildlife Conservation Policies",numberOfDownloads:233,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"May 15th 2020",dateEndSecondStepPublish:"June 5th 2020",dateEndThirdStepPublish:"August 4th 2020",dateEndFourthStepPublish:"October 23rd 2020",dateEndFifthStepPublish:"December 22nd 2020",remainingDaysToSecondStep:"9 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Dr. Kideghesho served as a Deputy Director of Wildlife Division in Tanzania's Ministry of Natural Resources and Tourism for two years (2012-2014). 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(Agriculture) from Sokoine University of Agriculture (SUA), Tanzania in 1993. He has worked for over 25 years in various positions in the Wildlife Sector as Assistant Lecturer at CAWM, Mweka, Lecturer, Senior Lecturer and Associate Professor at the Department of Wildlife Management at SUA and Assistant Director (Wildlife Utilization) in the Ministry of Natural Resources and Tourism. Kideghesho has been involved in over 20 local and international professional committees and organizations as a member, moderator, external examiner and a reviewer of scientific journals. He has published about 60 scientific articles and book chapters in peer reviewed journals and edited books, several articles in Conference Proceedings and 9 in Tanzania’s popular Wildlife Magazine. He is an editor of a book titled - Wildlife Management: Failures, Successes and Prospects. 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L. chagasi), an intracellular parasite of the phagocytic mononuclear system [1, 2]. In Brazil, VL is transmitted by sandflies, Lutzomyia longipalpis [1, 3, 4].
\nIn a global scenario, it is estimated that 300,000 new cases of VL occur with a rate of 20,000 deaths each year, with 94% new cases reported in Brazil, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan [5]. While in Latin America, LV spreads from Mexico to Argentina, with the largest number of cases concentrated in Brazil [6]. With the urbanization of VL in Brazil, annually, the country records approximately 3500 new cases, mainly in medium and large cities; probably, it is due to the disordered anthropic occupation of the geographic space [7]. Despite scientific advances, cases of VL are expanding, which has a major impact on public health, as dogs are the main reservoirs in the urban environment and therefore play an important role in the transmission cycle [8, 9].
\nCanine visceral leishmaniasis (CVL) is characterized by a broad clinical spectrum, from mild and moderate to fatal clinical manifestations. Major clinical signs in dogs include hepatosplenomegaly, lymphadenopathy, exfoliative dermatitis, alopecia, onychogryphosis, keratoconjunctivitis, apathy, anorexia, and severe weight loss [10, 11, 12, 13].
\nThe clinical manifestation of CVL depends on the interaction of the parasite with the host immune response [2]. In susceptible dogs, clinicopathological abnormalities are preceded by an evident humoral response and depression of the cellular response, mediated by a non-protective Th2 immune response associated with cytokines IL-4, IL-5, IL-6, and IL-10 [14, 15]. Dogs that do not develop the disease have a protective cellular response (Th1) [16, 17], related to INF-γ, TNF-α, IL-2, and IL-12 cytokines.
\nDifferent procedures are used for the diagnosis of CVL [18]. The Brazilian Ministry of Health recommends serology in the investigation of canine disease by the Dual-Path Platform (DPP®) rapid method as a screening test and ELISA as confirmatory test [19]. Other tests are used to demonstrate infection, such as cytology, histopathology [20], and real-time PCR (RT-PCR) [21].
\nSimilarly, determination of parasitic index has become important for early detection, but also evaluation of treatment efficacy and monitoring of relapses [22]. Thus, the aim of this study was to associate parasitic index to serum cytokine concentration in dogs naturally infected by L. infantum at different clinical stages of infection.
\nThe procedures were previously approved by the Ethics Committee on the Use of Animals (ECUA)/UFMT, Brazil (n° 23108.019567/14-1), and collection of clinical samples was authorized by the dog owners by signing the informed consent form.
\nThis study was conducted over a 16-month period, evaluating 42 male and female dogs of different ages and breeds from Barra do Garças, Mato Grosso State, Brazil (latitude, −15.893; longitude, 52.2599; south,15° 53′ 35″; west 52° 15′ 36″). Dogs with canine visceral leishmaniasis (n = 21) were classified into clinical stages at diagnosis as described by Solano et al. [23] and confirmed using the Dual-Path Platform Rapid Test (RT DPP®) and polymerase chain reaction (PCR). A control group (n = 21) was also formed, comprising dogs with no clinical changes and negative results for RT DPP® and conventional PCR.
\nBlood samples (5 mL) were collected by cephalic or jugular venipuncture, placed in tubes without anticoagulant to obtain serum. Serum was obtained by centrifuging the blood sample at 300× g for 5 minutes and was then transferred to 2 mL microtubes and stored at −80°C for cytokine dosing.
\nAfter dog restraint and local anesthesia with 2% lidocaine, bone marrow samples were obtained from the sternal manubrium, placed in microtubes with 0.5 mL 0.9% sterile NaCl solution, and stored at −20°C for subsequent molecular techniques.
\nThe immunochromatographic rapid test for detection of anti-Leishmania infantum antibodies (DPP®—Canine Visceral Leishmaniasis-Bio-Manguinhos/FIOCRUZ, Rio de Janeiro, Brazil) that uses the recombinant protein K39 (rK39) as an antigen, a cloned 39 amino acid sequence of the specific L. infantum kinase region, was performed according to the manufacturer’s guidance.
\nDNA extraction from blood samples was performed by the phenol-chloroform method. The polymerase chain reaction assay was performed using the primers RV1 (sense) 5′-CTT TTC TGG TCC CGC GGG TAG G-3′ and RV2 (antisense) 5′-CCA CCT GGC TAT TTT ACA CCA-3′ [24], which amplifies the DNA fragment of a 145 bp region of conserved kDNA present in L. infantum. Amplification used 200 mM dNTP, 1 pM from each primer, a buffer solution (10 mM Tris–HCl and 50 mM KCl, pH 8.3), 2 mM MgCl2, 1.5 U Taq DNA polymerase, and 2 μl of the DNA sample in the final volume of 25 μl. Assays were performed for one cycle at 94°C for 4 minutes, followed by 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and final extension of one cycle at 72°C for 10 minutes. The amplification product was fractionated by 2.0% agarose gel electrophoresis, stained with red gel spot, and visualized on a transilluminator (UV, 300 nm).
\nQuantitative PCR (qPCR) was performed in triplicate using the StepOne™ Real-Time PCR System Sequence Detection System (Applied Biosystems) targeting RV1–5′-CTT TTC TGG TCC GGG TAG G-3′ primers and RV2–5′-CCA CCT GGC TAT TTT ACA CCA-3′ amplifying a 145 bp sequence of L. infantum-specific kDNA [24]. Reactions were prepared in a 25 μl final volume containing SYBR Green Master Mix, 0.3 μM of each primer, and 2 μl of target DNA. Amplification conditions included an initial incubation step at 94°C for 10 minutes, followed by 40 cycles of amplification, 94°C for 15 seconds, and 60°C for 60 seconds. The standard curve was established for each assay using known amounts of TOPO PCR 2.1 plasmid (Invitrogen Corp.) containing L. infantum kDNA gene. Serial (10×) dilutions of the recombinant plasmid containing 2.9×104–2.9×108 copies of the plasmid were performed and used on the standard curve.
\nSerum cytokine concentration (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was assessed using the Cytometric Bead Array (CBA) Kit (BD Bioscience, USA) and evaluated by a flow cytometer (FACSCalibur®, BD Bioscience, USA). The reading was done using the CellQuest. Data were analyzed in FCAP array software version 5.0.
\nImmunoglobulin concentrations (IgM and IgG) in the sera were determined by turbidimetric method. For 1:11 (v/v) IgM and 1:15 (v/v) IgG, antibody concentrations were determined using IgM (Bioclin®, Brazil, Ref K063) and IgG (Bioclin®, Brazil, Ref K062) antiserum diluted with 1:12 (v/v). The calibration curve obtained from the Multical calibrator (Bioclin®, Brazil, Ref K064) was used to determine the standard curve for each immunoglobulin. Positive and negative serum samples, standards, and controls were placed in 500 μl buffer solution (0.15 mol/L sodium chloride, Tris 50 mmol/L, 6.0000 PEG 50 g/L, and sodium azide 15.38 nmol/L). The suspensions were mixed and incubated at 37°C for 10 minutes. Reactions were read on a spectrophotometer at 340 nm.
\nFor the analysis of the concentration of cytokines and immunoglobulins (IgG and IgM), the Student t-test independent samples were used. For the quantification of parasitic index of the bone marrow and cytokines when compared by clinical stage, Kruskal-Wallis analysis of variance was used. Parasite load correlation analysis of IgG in the presence of cytokines was also performed by calculating the Spearman correlation coefficient. Data were expressed as mean ± standard error. Values less than 0.05 (p < 0.05) were considered significant.
\nMost of the 21 dogs in the control group were mongrel dog (15/71%), Labrador retriever (1/5%), dachshund (1/5%), pinscher (3/14%), and rottweiler (1/5%). Age ranged from 14 months to 8 years (average 3.4 years). Thirteen dogs were female (13/62%) and eight dogs were male (8/38%). Most of the 21 dogs with leishmaniasis were dogs from mongrel dog (12/57%), Labrador retriever (1/05%), American pit bull (1/05%), poodle (1/05%), and shih tzu (6/28%). Age ranged from 12 months to 11 years (mean 4.3 years). Six dogs were female (6/29%) and 15 dogs were male (15/72%).
\nAt the time of clinical evaluation, all dogs diagnosed with VL had several clinicopathological findings typical of the disease. Clinical symptoms in seropositive animals (CVL) included lymphadenopathy (17/13%), skin ulcers (12/10%), onychogryphosis (11/09%), ear ulceration (11/09%), scaling (10/08%), weight loss (9/07%), dermatopathy (8/06%), ophthalmopathy (8/06%), muscle atrophy (4/03%), splenomegaly (7/06%), alopecia (6/05%), lethargy (5/04%), periocular alopecia (4/03%), skin nodules (3/02%), hepatomegaly (3/02%), cachexia (3/02%), and hyperkeratosis (2/01%).
\nDogs were classified into three clinical stages: stage I, mild disease (n = 5/24%); stage II, moderate disease (n = 9/43%); and stage III, severe disease (n = 7/33%). Stage II dogs were not subclassified.
\n\nLeishmania infantum DNA was detected in all dogs of the group with CVL up to a concentration of 1 fg/μl. Real-time PCR of bone marrow samples was positive in all dogs in the CVL group (100%). There was no statistical difference in the distribution between clinical stages and parasitic index, as shown in Table 1.
\nCytokines/parasitemia | \nI | \nII | \nIII | \np-Value | \n
---|---|---|---|---|
IL-2 | \n6.62 ± 1.18 | \n12.01 ± 7.99 | \n15.09 ± 6.34 | \n0.152 | \n
IL-4 | \n10.50 ± 2.05 | \n11.38 ± 3.81 | \n9.90 ± 2.73 | \n0.9044 | \n
IL-6 | \n2.14 ± 0.57 | \n2.72 ± 0.66 | \n3.12 ± 0.50 | \n0.0350 | \n
IL-10 | \n2.47 ± 0.97 | \n2.85 ± 0.96 | \n2.39 ± 0.84 | \n0.8973 | \n
IL-17 | \n2.22 ± 0.22 | \n12.38 ± 9.63 | \n13.27 ± 7.51 | \n0.4345 | \n
TNF-α | \n4.52 ± 2.12 | \n4.65 ± 2.31 | \n6.14 ± 1.43 | \n0.0462 | \n
IFN | \n3.07 ± 0.99 | \n28.19 ± 23.21 | \n2.58 ± 0.28 | \n0.4648 | \n
Parasite copy number (×107)/ml | \n4.96 ± 1.00 | \n4.63 ± 1.37 | \n4.55 ± 1.49 | \n0.9467 | \n
Cytokine concentrations and parasite copy number (×107)/ml in dogs with visceral leishmaniasis in different clinical staging.
The results were expressed in mean and standard error.
The mean and standard error of concentrations (pg/ml) of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17 cytokines based on clinical staging in CVL-infected dogs are shown in Table 1. It was observed that IL-6 and TNF-α concentrations increased in serum of infected dogs with significant statistical difference between the clinical stages of CVL, although most infected dogs had moderate and severe clinical manifestations of the disease.
\nAmong dogs with CVL and uninfected dogs, an increase of IL-4 and TNF-α concentrations in serum from dogs infected with CVL was observed. Similar serum concentrations of IL-2, IL-10, IL-17, and IFN-γ were observed between the groups studied (Table 2).
\nCytokines | \nControl | \nCVL | \np-Value | \n
---|---|---|---|
IL-2 | \n9.18 ± 6.14 | \n11.75 ± 6.89 | \n0.3199 | \n
IL-4 | \n7.43 ± 2.50 | \n12.56 ± 5.37 | \n0.0469 | \n
IL-6 | \n2.87 ± 0.95 | \n2.71 ± 0.67 | \n0.3326 | \n
IL-10 | \n2.98 ± 1.39 | \n2.62 ± 0.87 | \n0.2807 | \n
IL-17 | \n11.12 ± 12.12 | \n11.63 ± 9.66 | \n0.4570 | \n
TNF-α | \n2.80 ± 0.52 | \n5.12 ± 2.33 | \n0.0009 | \n
IFN | \n13.26 ± 16.88 | \n16.15 ± 19.01 | \n0.3589 | \n
Cytokine concentrations in dogs noninfected and dogs with canine visceral leishmaniasis.
The results were expressed in mean and standard error.
When comparing immunoglobulin means, IgG levels were elevated in the CVL group when compared to IgM levels. A significant difference (p = <0.0001) was observed. Similarly, IgG concentration between the control and CVL groups was evaluated. IgG levels were found to be higher in serum from dogs with CVL (2300.75 ± 678.463) when compared to control group IgG concentrations (636.94 ± 312.8 mg/dl), showing a significant difference between groups (p = <0.0001). Regarding the comparison of IgM concentration (mg/dl) in the CVL group (279.74 ± 37.755) compared to the control group (241.12 ± 59.835), there was no difference (Table 3).
\nGroup | \nControl | \nCVL | \np-Value | \n
---|---|---|---|
IgG | \n636.94 ± 255.52 | \n2288.04 ± 610.08 | \n<0.0001 | \n
IgM | \n241.12 ± 51.81 | \n282.42 ± 33.99 | \n0.0773 | \n
Immunoglobulin concentrations (IgG and IgM) in serum from dogs with canine visceral leishmaniasis.
The results were expressed in mean and standard error.
Correlations of IL-6 and TNF-α concentrations were analyzed according to clinical staging with parasitic index according to stage I, IL-6 (rs = 0.400, p = 0.5046) and TNF-α (rs = 0.700, p = 0.1881); stage II, IL-6 (rs = 0.7000, p = 0.1881) and TNF-α (rs = −0.1590, p = 0.6828); and stage III, IL-6 (rs = −0.3571, p = 0.4316) and TNF-α (rs = −0.4643, p = 0.2939). There was no correlation between the other parameters evaluated.
\nThe correlation between the parasitic index of dogs with CVL in the presence of cytokine IL-4 and TNF-α in the blood of dogs infected with CVL presented the IL-4 (rs = 0.0240, p = 0.9176) and TNF-α (rs = 0.0825, p = 0.7221). No additional significant correlations were found. Antibody levels were positively correlated with IL-4 expression (rs = 0.5997, p = 0.0040) (Table 4).
\n\n | IgG | \n\n |
---|---|---|
Parameters | \nrs | \np-Value | \n
IL-4 | \n0.5997 | \n0.0040 | \n
TNF-α | \n0.4164 | \n0.0603 | \n
Parasitic index | \n−0.2243 | \n0.3282 | \n
Correlation between IgG concentrations with IL-4 and TNF-α and parasitic index of dogs infected with CVL.
rs, correlation coefficient of Spearman.
In this study, as shown in Table 5, the correlation of the evolution of clinical signs between the stages presented below was analyzed. There was a significant positive correlation of IL-6 cytokine levels between stage I and stage III.
\nStage | \nI and II | \nI and III | \nII and III | \n|||
---|---|---|---|---|---|---|
\n | rs | \np | \nrs | \np | \nrs | \np | \n
IL-6 | \n0.6031 | \n0.0855 | \n0.8469 | \n0.0162 | \n0.5630 | \n0.1144 | \n
TNF-α | \n0.0350 | \n0.9288 | \n0.3784 | \n0.4026 | \n0.0168 | \n0.9658 | \n
Correlation of IL-6 and TNF-α cytokine levels of dogs with canine visceral leishmaniasis by clinical staging of serum from dogs of the CVL group.
rs, correlation coefficient of Spearman.
In this study the most dogs in the control group and CVL were mixed breed. The clinical symptoms of seropositive dogs (CVL) included lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, scaling, weight loss, and others. Dogs were classified into three clinical stages: stage I, mild disease; stage II, moderate disease; and stage III, severe disease. There was no statistical difference in the distribution between clinical stages and parasitic index. IL-6 and TNF-α concentrations increased in serum from infected dogs with a statistically significant difference between the clinical stages of CVL. Between the dogs with CVL and the control group, there was a statistical difference in the serum concentrations of cytokines IL-4 and TNF-α. IgG levels were elevated in the CVL group when compared to IgM levels. Antibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). There was a significant positive correlation of IL-6 cytokine levels between stage I and stage III.
\nThe clinical signs of CVL are important for the diagnosis. In the present study, the most prevalent clinical signs were lymphadenopathy, skin ulcers, onychogryphosis, ear ulceration, and scaling. However, prevalence is highly variable across studies, but generally these clinical signs are the most commonly reported in the literature. These results corroborate the findings of several authors [25, 26].
\nRegarding gender, there was a greater predominance of males in infected dogs and females in dogs in the control group. Regarding age, it did not present large variations. This fact seems to be associated with the higher risk of male exposure. However, the study shows no statistically significant differences for age and gender between healthy and sick dogs [27].
\nBone marrow samples were taken from 21 dogs serologically positive for L. infantum. According to the clinical signs, dogs were classified as stages I, II, and III. Real-time PCR detected no parasite copies (×1010)/μl L. infantum DNA in all animals of the CVL group, distributed as follows: stage I mean (4.964), stage II average (4.63), and stage III (4.55). No statistically significant difference was found in the average amount of DNA copy number between the different clinical stages (p = 0.9467). In bone marrow samples from dogs that are cytologically positive, a high parasitic index is detected [21].
\nPrevious studies report that quantitative PCR on bone marrow samples from positive dogs in conventional tests contained a higher number of Leishmania kDNA copies than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load [28]. This literary quote converges with the findings of this study.
\nPCR can be used for detection of Leishmania in naturally infected dog samples, and PCR-RFLP (restriction fragment length polymorphism) is sensitive for identification of Leishmania species [28]. In addition, qPCR is effective in quantifying Leishmania DNA loading in clinical samples [29]. The blood sample from dogs infected by L. infantum was found by real-time PCR to have a sensitivity of 100% and specificity of 96.4% [30].
\nMost cytokines remain partially conserved between species; in this sense, the amino acid sequence of humans and canine cytokines shows 49–96% homology, suggesting a high probability of cross-reactivity between monoclonal antibodies; thus antibodies against human cytokines may be recommended as immunological biomarkers under pathological conditions by flow cytometry in human [31] and dogs [32] as used in this study.
\nIn the present work, the serum concentration of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17) was compared between the control groups and the group with CVL. In addition, cytokine levels were compared within the CVL group with clinical staging I, II, and III. When comparing the groups, IL-4 and TNF-α were higher in infected dogs than in the control group, showing significant difference between IL-4 (p = 0.0469) and TNF-α (p = 0.0009) groups. In the group with CVL there were differences between stages I and III with significant differences only for cytokines IL-6 (p = 0.0350) and TNF-α (p = 0.0462).
\nElevated levels of IL-6 were found in serum from dogs with active leishmaniasis compared to healthy dogs [33]. These results corroborate the findings of this study. However, other authors reported that IL-6 production did not vary significantly between the groups studied [34]. On the other hand were described in the literature that elevated levels of IL-6 in dogs without clinical signs or symptoms in CVL dogs [35], and also highlights that, among other factors, it may indicate a balance between the parasite elimination effort and the active disease. Increased IL-6 levels suggest a restricted ability to control infection [36]. Even in the absence of clinical signs or symptoms, the animals showed granulomas on histopathological evaluation, suggesting chronicity and therefore a longtime course of infection [35]. Innate immune effector cells primarily neutrophils, monocytes, and macrophages produce and respond to IL-6, which may result in amplification of inflammation and a change from an acute inflammatory state to a chronic state [37].
\nIL-6 expression increases in dogs with active visceral leishmaniasis and may be a useful marker for active disease [33, 35]. Increased IL-6 production is not directly related to anti-Leishmania antibody titers, suggesting that other cytokines may be involved with hypergammaglobulinemia [33].
\nAs shown in this work, it was observed that there was correlation of IL-6 expression between stages I and III of bone marrow aspirate of dogs infected with CVL. IL-6 production in dogs with active leishmaniasis appears to be associated with severe disease [33]. This statement converges with the findings in this study, as the dogs used in the control group were mostly stage II and III. IL-6 is essential for terminal B-cell differentiation and immunoglobulin production [38].
\nTNF-α concentration was higher in infected dogs than in the control group, as detected by de Lima et al. [33]. CVL susceptibility is closely associated with downregulation of key cytokines such as IFN-γ, TNF-α, and IL-17A, thus impairing iNOS activation and NO production and favoring parasite replication and disease development [39].
\nThe increased activity of TNF-α in the liver of infected dogs compared to healthy canines has been reported [37, 40]. Higher TNF-α levels in infected dogs indicate that the presence of L. infantum induces an immune response with relevant TNF-α expression when the protozoan is present [40].
\nStudies suggest that decreased survival of L. infantum in canine macrophages is associated with increased TNF-α and IFN-γ production and decreased IL-10 production [41].
\nIn dogs naturally infected with L. infantum, increased hepatic TNF-α may be associated with increased parasite load on this organ [42]. The cytokines IL-2, IL-4, IL-10, IFN-γ, TNF-α, and IL-12 may be used as markers in epidemiological studies conducted in endemic areas to distinguish between different clinical forms of VL [15]. However, Lima et al. [33] indicate that TNF-α is not considered a good marker of active disease in dogs with VL.
\nA study has reported a significant relationship between bone marrow IL-4 detection in naturally infected dogs with and without clinical signs and disease severity, suggesting that IL-4 production is associated with pathology [43]. Increased expression of IL-4 cytokine is associated with both severe clinical signs and a high parasitic index on skin lesions [44]. In bone marrow aspirates, IL-4 was elevated in naturally infected dogs with more severe symptoms [43].
\nThe study points to evidence that IL-4 cytokine polymorphism may contribute to innate immunity to L. infantum infection [45].
\nAntibody levels were positively correlated with IL-4 expression (rs = 0.5997; p = 0.0040). IgG is also linked to chronic infection in patients with VL, where high levels of IgG are predictive of the disease. This finding is in line with the study by Lima et al. [33] suggesting that other cytokines, such as IL-10 or IL-4, may be associated with hypergammaglobulinemia observed in dogs with CVL. Previous studies have detected increased serum IgG levels in symptomatic dogs compared with healthy dogs and are related to pathophysiological disorders and active disease [33].
\nResponse to natural infection of L. infantum is linked to the presence of IgG [43] and Leishmania-specific IgM antibodies that can be detected in infected dogs [46]. Some studies have reported that increased total protein is frequent in dogs infected with visceral leishmaniasis due to increased antibody production [47, 48].
\nThese results may contribute to a better understanding of the immune response in dogs infected with L. infantum. Antibody levels were positively correlated with IL-4 expression. There was a significant positive correlation of IL-6 cytokine levels with the evolution of stages I and III. However, this cytokine can be used as a marker to distinguish between different clinical stages.
\nThis research received grants from the Mato Grosso Research Support Foundation (FAPEMAT No. 299032/2010) and from the National Council for Scientific and Technological Development (CNPq No. 447218/2014–0 and No. 305725/2018–1), in Brazil.
\nThe authors declare that there is no conflict of interest and nonfinancial competitors.
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\\n\\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\\n\\n7. MISCELLANEOUS
\\n\\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\\n\\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\\n\\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\\n\\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\\n\\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\\n\\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\\n\\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\\n\\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\\n\\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
\\n\\nLast updated: 2020-11-27
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The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
\n\n1. DEFINITIONS
\n\nCorresponding Author: The Author of the Chapter who serves as a Signatory to this Agreement. The Corresponding Author acts on behalf of any other Co-Author.
\n\nCo-Author: All other Authors of the Chapter besides the Corresponding Author.
\n\nIntechOpen: IntechOpen Ltd., the Publisher of the Book.
\n\nBook: The publication as a collection of chapters compiled by IntechOpen including the Chapter. Chapter: The original literary work created by Corresponding Author and any Co-Author that is the subject of this Agreement.
\n\n2. CORRESPONDING AUTHOR'S GRANT OF RIGHTS
\n\n2.1 Subject to the following Article, the Corresponding Author grants and shall ensure that each Co-Author grants, to IntechOpen, during the full term of copyright and any extensions or renewals of that term the following:
\n\nThe aforementioned licenses shall survive the expiry or termination of this Agreement for any reason.
\n\n2.2 The Corresponding Author (on their own behalf and on behalf of any Co-Author) reserves the following rights to the Chapter but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Chapter as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\n\nThe Corresponding Author confirms that they (and any Co-Author) are and will remain a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\n\nSubject to the license granted above, copyright in the Chapter and all versions of it created during IntechOpen's editing process (including the published version) is retained by the Corresponding Author and any Co-Author.
\n\nSubject to the license granted above, the Corresponding Author and any Co-Author retains patent, trademark and other intellectual property rights to the Chapter.
\n\n2.3 All rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the Corresponding Author's or any Co-Author’s specific approval.
\n\n2.4 The Corresponding Author (on their own behalf and on behalf of each Co-Author) will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Chapter as a consequence of IntechOpen's changes to the Chapter arising from translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits.
\n\n3. CORRESPONDING AUTHOR'S DUTIES
\n\n3.1 When distributing or re-publishing the Chapter, the Corresponding Author agrees to credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen. The Corresponding Author warrants that each Co-Author will also credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Chapter.
\n\n3.2 When submitting the Chapter, the Corresponding Author agrees to:
\n\nThe Corresponding Author will be held responsible for the payment of the Open Access Publishing Fees.
\n\nAll payments shall be due 30 days from the date of the issued invoice. The Corresponding Author or the payer on the Corresponding Author's and Co-Authors' behalf will bear all banking and similar charges incurred.
\n\n3.3 The Corresponding Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Chapter worldwide for the full term of the above licenses, and shall provide to IntechOpen upon request the original copies of such consents for inspection (at IntechOpen's option) or photocopies of such consents.
\n\nThe Corresponding Author shall obtain written informed consent for publication from people who might recognize themselves or be identified by others (e.g. from case reports or photographs).
\n\n3.4 The Corresponding Author and any Co-Author shall respect confidentiality rights during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Corresponding Author and any Co-Author are confidential and are intended only for the recipient. The contents may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\n\n4. CORRESPONDING AUTHOR'S WARRANTY
\n\n4.1 The Corresponding Author represents and warrants that the Chapter does not and will not breach any applicable law or the rights of any third party and, specifically, that the Chapter contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy. The Corresponding Author warrants and represents that: (i) the Chapter is the original work of themselves and any Co-Author and is not copied wholly or substantially from any other work or material or any other source; (ii) the Chapter has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) they themselves and any Co-Author are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) they themselves and any Co-Author have not assigned and will not during the term of this Publication Agreement purport to assign any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\n\nThe Corresponding Author also warrants and represents that: (i) they have the full power to enter into this Publication Agreement on their own behalf and on behalf of each Co-Author; and (ii) they have the necessary rights and/or title in and to the Chapter to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licenses expressed to be granted in this Publication Agreement. If the Chapter was prepared jointly by the Corresponding Author and any Co-Author, the Corresponding Author warrants and represents that: (i) each Co-Author agrees to the submission, license and publication of the Chapter on the terms of this Publication Agreement; and (ii) they have the authority to enter into this Publication Agreement on behalf of and bind each Co-Author. The Corresponding Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each such Co-Author.
\n\nThe Corresponding Author agrees to indemnify and hold IntechOpen harmless against all liabilities, costs, expenses, damages and losses and all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of or in connection with any breach of the aforementioned representations and warranties. This indemnity shall not cover IntechOpen to the extent that a claim under it results from IntechOpen's negligence or willful misconduct.
\n\n4.2 Nothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\n\n5. TERMINATION
\n\n5.1 IntechOpen has a right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Corresponding Author or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Corresponding Author or any Co-Author (being an individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Corresponding Author or any Co-Author (being a company) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for or enters into any compromise or arrangement with any of its creditors.
\n\nIn case of termination, IntechOpen will notify the Corresponding Author, in writing, of the decision.
\n\n6. INTECHOPEN’S DUTIES AND RIGHTS
\n\n6.1 Unless prevented from doing so by events outside its reasonable control, IntechOpen, in its discretion, agrees to publish the Chapter attributing it to the Corresponding Author and any Co-Author.
\n\n6.2 IntechOpen has the right to use the Corresponding Author’s and any Co-Author’s names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Chapter and has the right to contact the Corresponding Author and any Co-Author until the Chapter is publicly available on any platform owned and/or operated by IntechOpen.
\n\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\n7. MISCELLANEOUS
\n\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\n\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\n\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\n\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\n\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
\n\nLast updated: 2020-11-27
\n\n\n\n
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. I have served as the editor for many books, been a member of the editorial board in science journals, have published many papers and hold many patents.",institutionString:null,institution:{name:"Sheffield Hallam University",country:{name:"United Kingdom"}}},{id:"54525",title:"Prof.",name:"Abdul Latif",middleName:null,surname:"Ahmad",slug:"abdul-latif-ahmad",fullName:"Abdul Latif Ahmad",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"20567",title:"Prof.",name:"Ado",middleName:null,surname:"Jorio",slug:"ado-jorio",fullName:"Ado Jorio",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universidade Federal de Minas Gerais",country:{name:"Brazil"}}},{id:"47940",title:"Dr.",name:"Alberto",middleName:null,surname:"Mantovani",slug:"alberto-mantovani",fullName:"Alberto Mantovani",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"12392",title:"Mr.",name:"Alex",middleName:null,surname:"Lazinica",slug:"alex-lazinica",fullName:"Alex Lazinica",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/12392/images/7282_n.png",biography:"Alex Lazinica is the founder and CEO of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. Focus of his research activity is drug delivery, physico-chemical characterization and biological evaluation of biopolymers micro and nanoparticles as modified drug delivery system, and colloidal drug carriers (liposomes, nanoparticles etc.).",institutionString:null,institution:{name:"Marmara University",country:{name:"Turkey"}}},{id:"61051",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"100762",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"St David's Medical Center",country:{name:"United States of America"}}},{id:"107416",title:"Dr.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas Cardiac Arrhythmia",country:{name:"United States of America"}}},{id:"64434",title:"Dr.",name:"Angkoon",middleName:null,surname:"Phinyomark",slug:"angkoon-phinyomark",fullName:"Angkoon Phinyomark",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/64434/images/2619_n.jpg",biography:"My name is Angkoon Phinyomark. I received a B.Eng. degree in Computer Engineering with First Class Honors in 2008 from Prince of Songkla University, Songkhla, Thailand, where I received a Ph.D. degree in Electrical Engineering. My research interests are primarily in the area of biomedical signal processing and classification notably EMG (electromyography signal), EOG (electrooculography signal), and EEG (electroencephalography signal), image analysis notably breast cancer analysis and optical coherence tomography, and rehabilitation engineering. I became a student member of IEEE in 2008. During October 2011-March 2012, I had worked at School of Computer Science and Electronic Engineering, University of Essex, Colchester, Essex, United Kingdom. In addition, during a B.Eng. I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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