Chagas Disease: A Silent Threat for Dogs and Humans.

Chagas disease (CD) is a vector-borne Neglected Zoonotic Disease (NZD) caused by a flagellate protozoan, Trypanosoma cruzi, that affects various mammalian species across America, including humans and domestic animals. However, due to an increase in population movements and new routes of transmission, T. cruzi infection is presently considered a worldwide health concern, no longer restricted to endemic countries. Dogs play a major role in the domestic cycle by acting very efficiently as reservoirs and allowing the perpetuation of parasite transmission in endemic areas. Despite the significant progress made in recent years, still there is no vaccine against human and animal disease, there are few drugs available for the treatment of human CD, and there is no standard protocol for the treatment of canine CD. In this review, we highlight human and canine Chagas Disease in its different dimensions and interconnections. Dogs, which are considered to be the most important peridomestic reservoir and sentinel for the transmission of T. cruzi infection in a community, develop CD that is clinically similar to human CD. Therefore, an integrative approach, based on the One Health concept, bringing together the advances in genomics, immunology, and epidemiology can lead to the effective development of vaccines, new treatments, and innovative control strategies to tackle CD.

Quantum Biochemistry Screening and In Vitro Evaluation of Leishmania Metalloproteinase Inhibitors.

Leishmanolysin, also known as major promastigote protease (PSP) or gp63, is the most abundant surface glycoprotein of Leishmania spp., and has been extensively studied and recognized as the main parasite virulence factor. Characterized as a metalloprotease, gp63 can be powerfully inactivated in the presence of a metal chelator. In this study, we first used the structural parameters of a 7-hydroxycoumarin derivative, L1 compound, to evaluate the theoretical-computational experiments against gp63, comparing it with an available metal chelator already described. The methodology followed was (i) analysis of the three-dimensional structure of gp63 as well as its active site, and searching the literature and molecular databases for possible inhibitors; (ii) molecular docking simulations and investigation of the interactions in the generated protein-ligand complexes; and (iii) the individual energy of the gp63 amino acids that interacted most with the ligands of interest was quantified by ab initio calculations using Molecular Fraction with Conjugated Caps (MFCC). MFCC still allowed the final quantum balance calculations of the protein interaction to be obtained with each inhibitor candidate binder. L1 obtained the best energy quantum balance result with -2 eV, followed by DETC (-1.4 eV), doxycycline (-1.3 eV), and 4-terpineol (-0.6 eV), and showed evidence of covalent binding in the enzyme active site. In vitro experiments confirmed L1 as highly effective against L. amazonensis parasites. The compound also exhibited a low cytotoxicity profile against mammalian RAW and 3T3 cells lines, presenting a selective index of 149.19 and 380.64 µM, respectively. L1 induced promastigote forms' death by necrosis and the ultrastructural analysis revealed disruption in membrane integrity. Furthermore, leakage of the contents and destruction of the parasite were confirmed by Spectroscopy Dispersion analysis. These results together suggested L1 has a potential effect against L. amazonensis, the etiologic agent of diffuse leishmaniasis, and the only one that currently does not have a satisfactory treatment.

Intracellular and extracellular effector activity of mouse neutrophils in response to cutaneous and visceral Leishmania parasites.

Neutrophils are short-lived phagocytic cells equipped with several receptors for pathogen recognition and phagocytosis and have intracellular and extracellular effector mechanisms that can inactivate pathogens. Leishmaniases are diseases caused by different species of Leishmania that mainly afflicts poorer populations of tropical and subtropical regions and immunocompromised individuals. Thus, the present study aims to investigate the effector response of murine neutrophils to species of Leishmania causing American cutaneous leishmaniasis and zoonotic visceral leishmaniasis by evaluating pattern recognition receptors (PRR) and intracellular and extracellular effector microbicide activity. When exposed to Leishmania parasites, mouse neutrophils produced superoxide, released enzymes in the extracellular space and generated neutrophil extracellular traps, although PRR gene expression is negatively regulated. L. infantum, L. guyanensis, and L. shawi inhibited enzymatic activity, whereas L. amazonensis reduced the emission of extracellular structures. These findings indicate that although neutrophils trigger several microbicide mechanisms, Leishmania parasites can manipulate extracellular effector mechanisms. The present study also provides evidence that neutrophils can internalize parasites by coiling phagocytosis.

Canine neutrophils cooperate with macrophages in the early stages of Leishmania infantum in vitro infection.

Leishmania infantum is the aetiological agent of human visceral leishmaniasis and canine leishmaniasis, both systemic and potentially fatal diseases. Polymorphonuclear neutrophils (PMN) are the first cells to phagocyte this parasite at the inoculation site, but macrophages (MØ) are the definitive host cells, ensuring parasite replication. The interaction between dog MØ, PMN and L infantum promastigotes was in vitro investigated. It was observed that promastigotes establish contact with blood monocyte-derived MØ mainly by the tip of the flagellum. These cells, that efficiently bind and internalize parasites, underwent major morphological changes, produced nitric oxide (NO) and released histone H1 in order to inactivate the parasite. Transfer of intracellular parasites from PMN to MØ was confirmed by flow cytometry, using L infantum expressing a green fluorescent protein. The interaction of MØ with L infantum-infected PMN lead to NO production and release of extracellular traps, which may contribute to parasite containment and inactivation. This study highlights for the first time the diversity of cellular and molecular events triggered by the interaction between canine PMN and MØ, which can promote a reduction of parasite burden in the early phase of L infantum infection.

A field evaluation of an isothermal DNA amplification assay for the detection of Theileria annulata infection in cattle.

A loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Theileria annulata infection in cattle. The results were compared with a real-time PCR used for the quantification of T. annulata parasitaemia. One hundred bovine blood samples from 16 cattle farms were tested with LAMP and real-time PCR, with T. annulata DNA being detected in 66% and 67% of the samples, respectively. The results showed that the LAMP assay detects a parasitaemia as low as 0.00025%, indicating a high analytical sensitivity of LAMP for clinical diagnosis of bovine theileriosis.

Exploiting Leishmania-Primed Dendritic Cells as Potential Immunomodulators of Canine Immune Response.

Dendritic cells (DCs) capture pathogens and process antigens, playing a crucial role in activating naïve T cells, bridging the gap between innate and acquired immunity. However, little is known about DC activation when facing Leishmania parasites. Thus, this study investigates in vitro activity of canine peripheral blood-derived DCs (moDCs) exposed to L. infantum and L. amazonensis parasites and their extracellular vesicles (EVs). L. infantum increased toll-like receptor 4 gene expression in synergy with nuclear factor κB activation and the generation of pro-inflammatory cytokines. This parasite also induced the expression of class II molecules of major histocompatibility complex (MHC) and upregulated co-stimulatory molecule CD86, which, together with the release of chemokine CXCL16, can attract and help in T lymphocyte activation. In contrast, L. amazonensis induced moDCs to generate a mix of pro- and anti-inflammatory cytokines, indicating that this parasite can establish a different immune relationship with DCs. EVs promoted moDCs to express class I MHC associated with the upregulation of co-stimulatory molecules and the release of CXCL16, suggesting that EVs can modulate moDCs to attract cytotoxic CD8+ T cells. Thus, these parasites and their EVs can shape DC activation. A detailed understanding of DC activation may open new avenues for the development of advanced leishmaniasis control strategies.

Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model.

Insights on Host-Parasite Immunomodulation Mediated by Extracellular Vesicles of Cutaneous Leishmania shawi and Leishmania guyanensis.

Leishmaniasis is a parasitic disease caused by different species of Leishmania and transmitted through the bite of sand flies vector. Macrophages (MΦ), the target cells of Leishmania parasites, are phagocytes that play a crucial role in the innate immune microbial defense and are antigen-presenting cells driving the activation of the acquired immune response. Exploring parasite-host communication may be key in restraining parasite dissemination in the host. Extracellular vesicles (EVs) constitute a group of heterogenous cell-derived membranous structures, naturally produced by all cells and with immunomodulatory potential over target cells. This study examined the immunogenic potential of EVs shed by L. shawi and L. guyanensis in MΦ activation by analyzing the dynamics of major histocompatibility complex (MHC), innate immune receptors, and cytokine generation. L. shawi and L. guyanensis EVs were incorporated by MΦ and modulated innate immune receptors, indicating that EVs cargo can be recognized by MΦ sensors. Moreover, EVs induced MΦ to generate a mix of pro- and anti-inflammatory cytokines and favored the expression of MHCI molecules, suggesting that EVs antigens can be present to T cells, activating the acquired immune response of the host. Since nano-sized vesicles can be used as vehicles of immune mediators or immunomodulatory drugs, parasitic EVs can be exploited by bioengineering approaches for the development of efficient prophylactic or therapeutic tools for leishmaniasis.

Parasitic Infection and Immunity-A Special Biomedicines Issue.

Infectious parasitic diseases that affect humans and animals remain a central health concern worldwide [...].

Zoonotic Visceral Leishmaniasis: New Insights on Innate Immune Response by Blood Macrophages and Liver Kupffer Cells to Leishmania infantum Parasites.

L. infantum is the aetiological agent of zoonotic visceral leishmaniasis (ZVL), a disease that affects humans and dogs. Leishmania parasites are well adapted to aggressive conditions inside the phagolysosome and can control the immune activation of macrophages (MØs). Although MØs are highly active phagocytic cells with the capacity to destroy pathogens, they additionally comprise the host cells for Leishmania infection, replication, and stable establishment in the mammal host. The present study compares, for the first time, the innate immune response to L. infantum infection of two different macrophage lineages: the blood macrophages and the liver macrophages (Kupffer cells, KC). Our findings showed that L. infantum takes advantage of the natural predisposition of blood-MØs to phagocyte pathogens. However, parasites rapidly subvert the mechanisms of MØs immune activation. On the other hand, KCs, which are primed for immune tolerance, are not extensively activated and can overcome the dormancy induced by the parasite, exhibiting a selection of immune mechanisms, such as extracellular trap formation. Altogether, KCs reveal a different pattern of response in contrast with blood-MØs when confronting L. infantum parasites. In addition, KCs response appears to be more efficient in managing parasite infection, thus contributing to the ability of the liver to naturally restrain Leishmania dissemination.

A Questionnaire-Based Survey on the Long-Term Management of Canine Leishmaniosis by Veterinary Practitioners.

Canine Leishmaniosis (CanL) is a chronic and potentially fatal disease. In economically disadvantaged regions, costs associated with long-term patient monitoring may determine that some owners decline veterinary follow-up of their dogs. This online, questionnaire-based survey aimed to assess how Portuguese veterinary practitioners perform long-term patient monitoring and recognize relapses. More than 50% of respondents reported that 50-100% of dog owners declared financial restraints. Hence, in these circumstances, most veterinary practitioners only performed clinical examination and serology. However, when owners did not declare financial restriction, other tests were additionally performed, such as renal and hepatic profiles, hemogram, serum protein electrophoresis and urine protein creatinine ratio. The mean number of exams performed when owners presented financial restraints was significantly lower than the number of exams performed without economic limitations. Most veterinary practitioners prescribed allopurinol ad aeternum or until disease remission and domperidone. CanL relapses were recognized by more than half of respondents "Always", through the reappearance or worsening of clinical signs, whereas about a quarter detected an increase in anti-Leishmania antibody levels and identified abnormalities in the serum protein electrophoresis profile. The relapse rate was higher in the Lisbon Metropolitan Area and north, the most economically favored regions of Portugal. This study confirms that owner financial restraints negatively influence veterinary follow-up and relapse recognition, ultimately compromising clinical decision making and favoring the maintenance of Leishmania infantum infection endemicity.

African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma brucei brucei Manipulate Host Mononuclear Cells.

African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by Glossina spp. (tsetse fly). Parasite introduction into mammal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a microbicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the current study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative immunity. TbEVs induce the differentiation of both M1- and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem to display opposite but complementary effects in the host, establishing a balance between parasite growth and controlled immune response, at least during the early phase of infection.

Human toxoplasmosis in Mozambique: gaps in knowledge and research opportunities.

Toxoplasmosis is a parasitic zoonotic disease caused by Toxoplasma gondii that afflicts humans worldwide and wild and domestic warm-blooded animals. In immunocompetent individuals, the acute phase of infection presents transient low or mild symptoms that remain unnoticed. In immunocompromised patients, T. gondii is a life-threatening opportunistic infection, which can result from the reactivation of latent infection or primary infection. Moreover, congenital toxoplasmosis, which results from the transplacental passage of tachyzoites into the fetus during a pregnant primary infection, can lead to miscarriage, stillbirth, or ocular and neurologic disease, and neurocognitive deficits in the newborns. Thus, the present review aims to address the current knowledge of T. gondii infection and toxoplasmosis in Africa and especially in Mozambique, stressing the importance of identifying risk factors and promote awareness among the health care providers and population, assessing the gaps in knowledge and define research priorities. In Mozambique, and in general in southern African countries, clinical disease and epidemiological data have not yet been entirely addressed in addition to the implications of T. gondii infection in immunocompetent individuals, in pregnant women, and its relation with neuropsychiatric disorders. The main gaps in knowledge in Mozambique include lack of awareness of the disease, lack of diagnostic methods in health facilities, lack of genetic data, and lack of control strategies.

Prognostic Factors and Life Expectancy in Canine Leishmaniosis.

Canine leishmaniosis (CanL) is a chronic and potentially fatal disease. The prognosis of CanL depends on the severity of the clinical signs and clinicopathological abnormalities presented by the dog at the time of diagnosis. This study aims to estimate the survival time of dogs with CanL, determining the prognostic value of different clinical and clinicopathological parameters. Medical records of 99 dogs diagnosed with CanL in five veterinary centers of the Alentejo region (Portugal) were examined retrospectively. The majority of dogs presented hyperproteinemia, moderate normocytic normochromic anemia, normal blood urea and creatinine levels and were classified as stage 1 according to the International Interest Society (IRIS) guidelines at the time of diagnosis. The severity of anemia, presence of concomitant infectious diseases at the time of diagnosis and the anti-Leishmania therapy were correlated with the survival time. The influence of renal dysfunction was evaluated by Receiver Operating Characteristic (ROC) curve and survival analysis. Survival analysis demonstrated that patients classified as IRIS 1 at the time of diagnosis survived more than four years, in contrast with dogs classified as IRIS 2 that survived around two and half years and dogs classified as IRIS 3-4 that survived around one month. IRIS stage deteriorated during the course of CanL in one third of the dogs and was the principal cause of death or euthanasia in a high proportion of animals. In some cases, dogs did not receive anti-Leishmania treatment or abandoned the veterinary follow-ups, which may have considerable repercussions for animal wellbeing and public health. This study reinforces the value of blood urea and creatinine levels as prognostic factors in CanL.

Immunophenotyping of Peripheral Blood, Lymph Node, and Bone Marrow T Lymphocytes During Canine Leishmaniosis and the Impact of Antileishmanial Chemotherapy.

Dogs are a major reservoir of Leishmania infantum, etiological agent of canine leishmaniosis (CanL) a zoonotic visceral disease of worldwide concern. Therapeutic protocols based on antileishmanial drugs are commonly used to treat sick dogs and improve their clinical condition. To better understand the impact of Leishmania infection and antileishmanial drugs on the dog's immune response, this study investigates the profile of CD4+ and CD8+ T cell subsets in peripheral blood, lymph node, and bone marrow of sick dogs and after two different CanL treatments. Two CanL groups of six dogs each were treated with either miltefosine or meglumine antimoniate combined with allopurinol. Another group of 10 clinically healthy dogs was used as control. Upon diagnosis and during the following 3 months of treatment, peripheral blood, popliteal lymph node, and bone marrow mononuclear cells were collected, labeled for surface markers CD45, CD3, CD4, CD8, CD25, and intracellular nuclear factor FoxP3, and T lymphocyte subpopulations were immunophenotyped by flow cytometry. CanL dogs presented an overall increased frequency of CD8+ and CD4+CD8+ double-positive T cells in all tissues and a decreased frequency of CD4+ T cells in the blood. Furthermore, there was a higher frequency of CD8+ T cells expressing CD25+FoxP3+ in the blood and bone marrow. During treatment, these subsets recovered to levels similar to those of healthy dogs. Nevertheless, antileishmanial therapy caused an increase of CD4+CD25+FoxP3+ T cells in all tissues, associated with the decrease of CD8+CD25-FoxP3- T cell percentages. These findings may support previous studies that indicate that L. infantum manipulates the dog's immune system to avoid the development of a protective response, ensuring the parasite's survival and the conditions that allow the completion of Leishmania life cycle. Both treatments used appear to have an effect on the dog's immune response, proving to be effective in promoting the normalization of T cell subsets.

3D-Hepatocyte Culture Applied to Parasitology: Immune Activation of Canine Hepatic Spheroids Exposed to Leishmania infantum.

The application of innovative three-dimensional (3D) spheroids cell culture strategy to Parasitology offers the opportunity to closely explore host-parasite interactions. Here we present a first report on the application of 3D hepatic spheroids to unravel the immune response of canine hepatocytes exposed to Leishmania infantum. The liver, usually considered a major metabolic organ, also performs several important immunological functions and constitutes a target organ for L. infantum infection, the etiological agent of canine leishmaniasis (CanL), and a parasitic disease of major veterinary and public health concern. 3D hepatic spheroids were able to sense and immunologically react to L. infantum parasites, generating an innate immune response by increasing nitric oxide (NO) production and enhancing toll-like receptor (TLR) 2 and interleukin-10 gene expression. The immune response orchestrated by canine hepatocytes also lead to the impairment of several cytochrome P450 (CYP450) with possible implications for liver natural xenobiotic metabolization capacity. The application of meglumine antimoniate (MgA) increased the inflammatory response of 3D hepatic spheroids by inducing the expression of Nucleotide oligomerization domain (NOD) -like receptors 1 and NOD2 and TLR2, TLR4, and TLR9 and enhancing gene expression of tumour necrosis factor α. It is therefore suggested that hepatocytes are key effector cells and can activate and orchestrate the immune response to L. infantum parasites.

Cutaneous Leishmaniasis: The Complexity of Host's Effective Immune Response against a Polymorphic Parasitic Disease.

This review is aimed at providing a comprehensive outline of the immune response displayed against cutaneous leishmaniasis (CL), the more common zoonotic infection caused by protozoan parasites of the genus Leishmania. Although of polymorphic clinical presentation, classically CL is characterized by leishmaniotic lesions on the face and extremities of the patients, which can be ulcerative, and even after healing can lead to permanent injuries and disfigurement, affecting significantly their psychological, social, and economic well-being. According a report released by the World Health Organization, the disability-adjusted life years (DALYs) lost due to leishmaniasis are close to 2.4 million, annually there are 1.0-1.5 million new cases of CL, and a numerous population is at risk in the endemic areas. Despite its increasing worldwide incidence, it is one of the so-called neglected tropical diseases. Furthermore, this review provides an overview of the existing knowledge of the host innate and acquired immune response to cutaneous species of Leishmania. The use of animal models and of in vitro studies has improved the understanding of parasite-host interplay and the complexity of immune mechanisms involved. The importance of diagnosis accuracy associated with effective patient management in CL reduction is highlighted. However, the multiple factors involved in CL epizoology associated with the unavailability of vaccines or drugs to prevent infection make difficult to formulate an effective strategy for CL control.

Development of Dog Immune System: From in Uterus to Elderly.

Immune system recognize and fight back foreign microorganisms and inner modifications that lead to deficient cell and tissue functions. During a dog's life, the immune system needs to adapt to different physiological conditions, assuring surveillance and protection in a careful and controlled way. Pregnancy alters normal homeostasis, requiring a balance between immunity and tolerance. The embryos and fetus should be protected from infections, while the female dog must tolerate the growing of semi-allografts in her uterus. After birth, newborn puppies are at great risk of developing infectious diseases, because their immune system is in development and immune memory is absent. Passive transfer of immunity through colostrum is fundamental for puppy survival in the first weeks of life, but hampers the development of an active immune response to vaccination. At the end of life, dogs experience a decline in the structure and functional competence of the immune system, compromising the immune responses to novel antigenic challenges, such as infections and vaccines. Therefore, the current article reviews the general processes related to the development of the dog´s immune system, providing an overview of immune activity throughout the dog's life and its implications in canine health, and highlighting priority research goals.

Meglumine Antimoniate and Miltefosine Combined With Allopurinol Sustain Pro-inflammatory Immune Environments During Canine Leishmaniosis Treatment.

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-ß, and IFN-γ by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-γ and downregulation of IL-2, IL-4, and TGF-ß, while gene expression of IL-12, TNF-α, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-γ gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.