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There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite., Competing Interests: The authors have declared that no competing interests exist.

Ticks are important ectoparasites which transmit many disease pathogen to animals; these are labelled tick borne diseases (TBD). Tick induced damage to skin and hides has not received attention. Skin and hides are important for the leather product industry, particularly in Pakistan. Due to economic importance and financial loss by ticks in leather industry, the present study was designed to investigate skin and hides damage due to ticks at microscopic level. Naturally tick infested tissue samples of hides and skin were collected from slaughter houses. Primary lesions at tick feeding sites showed epidermal edema with adjacent dermal edema. Histopathological examination revealed degeneration of epidermal layer down to the basal layer. Epidermal and sub dermal layers often displayed focal necrosis infiltrated with neutrophils and mononuclear cells at tick bite sites. Hyperplasia of keratinocytes was also seen at sites of ruptured epidermis. Quality of leather depends upon the grain (Outer) surface skin/hides. Ticks infestation damages the outer surface, due to bites, inflammatory responses, and secondary bacterial infections that often become established at feeding sites. Control of ticks should be given consideration to reduce infestation induced losses in the leather industry in Pakistan.

Lesion features in cutaneous leishmaniasis (CL) depend on the infecting Leishmania species as well as on host immune reponse. In this study, we evaluated the histological and immunological differences between two forms of CL described in Tunisia: zoonotic cutaneous leishmaniasis (ZCL) caused by L. major and sporadic cutaneous leishmaniasis (SCL) caused by L. infantum. Histological analysis showed a mild to moderate infiltrate within ZCL lesions. In contrast, massive infiltration of the dermis was observed within SCL lesions. Contrary to ZCL, infiltrates within SCL lesions were organized and showed granuloma composed of macrophages and lymphocytes. In addition, immunohistochemical analysis showed a predominance of CD4 + T cells within both CL forms. Furthermore, expression of interferon-γ, interleukin (IL)-10, IL-8, IL-13 and monocyte chemotactic protein (MCP)-1 was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR). MCP-1 and IL-10 were expressed at comparable levels in ZCL and SCL lesions. Interestingly, IL-8 mRNA levels were significantly higher in ZCL lesions compared to SCL lesions, but interferon-γ was significantly higher in SCL lesions than in ZCL lesions., (© T. Boussoffara et al., published by EDP Sciences, 2019.)

Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site. IMPORTANCE Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity., (Copyright © 2018 Flores-Garcia et al.)

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.

AbstractSeveral case reports of autochthonous leishmaniasis in Thailand have been published since 1996. Most of the previous cases presented with visceral leishmaniasis (VL) and were mostly reported in southern part of Thailand. Recently, it has been evident that Leishmania martiniquensis is the main cause of Leishmania infection in Thailand. However, Leishmania siamensis (PCM2 Trang isolate) was found to be of a separate lineage with restricted distribution in southern Thailand and also a cause of disseminated dermal and visceral leishmaniasis in one published case. Here we report the first patient from central Thailand with human immunodeficiency virus infection presenting with disseminated dermal leishmaniasis. Polymerase chain reaction and DNA sequencing analysis (large subunit of RNA polymerase II and 18S ribosomal RNA internal transcribed spacer 1) from the tissue biopsy sample revealed the pathogen sequences to be highly homologous to PCM2 Trang strain previously reported from southern Thailand.

American cutaneous leishmaniasis (ACL) is a chronic infectious disease caused by different protozoan species of Leishmania, and it is endemic in both tropical and subtropical countries. Using immunohistochemistry, we investigate the density of CD68 + , lysozyme + , CD1a + , factor XIIIa + , CD4 + , CD8 + , CD56 + , interferon (IFN)-γ + , and inducible NO synthase (iNOS + ) cells. These cells were analyzed from 22 biopsy samples obtained from the lesions of ACL patients, whose infection was caused by Leishmania (Viannia) spp. Histopathological analysis showed dense mononuclear inflammatory infiltration in the dermis, which was composed of lymphocytes, macrophages, plasma cells, and discrete tissue parasitism. Granulomatous reactions were also present in the majority of cases. The density of the activated macrophages was higher than that of inactivated macrophages in the lesions. The density of Langerhans cells (CD1a + ) was lower than that of dermal dendrocytes (factor XIIIa + ). The density of CD8 + T lymphocytes was higher than that of CD4 + T lymphocytes. The cellular density of these immunological markers in relation to the species of Leishmania demonstrated that L. (Viannia) sp. lesions had higher IFN-γ expression than that Leishmania (Viania) braziliensis lesions. The evaluation of these markers, according to disease progression, did not reveal any significant differences. L. (Viannia) sp. infection leads to a favorable immune response in the host, as predominantly represented by lysozyme + , factor XIIIa + , CD8 + T cells, and the expression of (IFN)-γ + at the lesion site.

Malaria is a pernicious infectious disease caused by apicomplexan parasites of the genus Plasmodium. Each year, malaria afflicts over 200million people, causing considerable morbidity, loss to gross domestic product of endemic countries, and more than 420,000 deaths. A central feature of the virulence of malaria parasites is the ability of sporozoite forms injected by a mosquito to navigate from the inoculation site in the skin through host tissues to infect the liver. The ability for sporozoites to traverse through different host cell types is very important for the successful development of parasites within the mammalian host. Over the past decade, our understanding of the role of host cell traversal has become clearer through important studies with rodent models of malaria. However, we still do not understand the stepwise process of host cell entry and exit or know the molecular mechanisms governing each step. We know even less about cell traversal by malaria parasite species that infect humans. Here, we review current knowledge regarding the role and molecular mechanisms of sporozoite cell traversal and highlight recent advances that prompt new ways of thinking about this important process., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Cutaneous leishmaniasis causes a spectrum of diseases from self-healing to severe nonhealing lesions. Defining the factors contributing to lesion resolution may help in developing new therapies for those patients with chronic disease. We found that infection with Leishmania major increases the expression of vascular endothelial growth factor-A and vascular endothelial growth factor receptor (VEGFR)-2 and is associated with significant changes in the blood and lymphatic vasculature at the site of infection. Ab blockade of VEGFR-2 during infection led to a reduction in lymphatic endothelial cell proliferation and simultaneously increased lesion size without altering the parasite burden. These data show that L. major infection initiates enhanced vascular endothelial growth factor-A/VEGFR-2 signaling and suggest that VEGFR-2-dependent lymphangiogenesis is a mechanism that restricts tissue inflammation in leishmaniasis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Tsetse flies are the sole vectors of Trypanosoma brucei parasites that cause sleeping sickness. Our knowledge on the early interface between the infective metacyclic forms and the mammalian host skin is currently highly limited. Glossina morsitans flies infected with fluorescently tagged T. brucei parasites were used in this study to initiate natural infections in mice. Metacyclic trypanosomes were found to be highly infectious through the intradermal route in sharp contrast with blood stream form trypanosomes. Parasite emigration from the dermal inoculation site resulted in detectable parasite levels in the draining lymph nodes within 18 hours and in the peripheral blood within 42 h. A subset of parasites remained and actively proliferated in the dermis. By initiating mixed infections with differentially labeled parasites, dermal parasites were unequivocally shown to arise from the initial inoculum and not from a re-invasion from the blood circulation. Scanning electron microscopy demonstrated intricate interactions of these skin-residing parasites with adipocytes in the connective tissue, entanglement by reticular fibers of the periadipocytic baskets and embedment between collagen bundles. Experimental transmission experiments combined with molecular parasite detection in blood fed flies provided evidence that dermal trypanosomes can be acquired from the inoculation site immediately after the initial transmission. High resolution thermographic imaging also revealed that intradermal parasite expansion induces elevated skin surface temperatures. Collectively, the dermis represents a delivery site of the highly infective metacyclic trypanosomes from which the host is systemically colonized and where a proliferative subpopulation remains that is physically constrained by intricate interactions with adipocytes and collagen fibrous structures.

In vivo imaging has revealed new details about how the malaria parasite enters the bloodstream.

Objective: Lernaeids are crustacean parasites that are globally distributed among freshwater and marine fish. Approximately 110 species of Lernaeids have been divided into 14 genera. The most common species of Lernaeids is Lernaea cyprinacea, which has been transmitted to ornamental fish worldwide. The economic importance of L. cyprinacea is increasing because of the epidemic caused by the parasite in most of the ornamental fish breeding centers in different parts of the world. The parasite affects its host's health, decreases growth rate, and causes abnormal metabolic activity. Accumulation of these parasites in some parts of the body causes painful points and has harmful outcomes for the functioning and survival of the host., Methods: The present study was conducted to examine the prevalence of L. cyprinacea among ornamental fish within 1 year from September-October 2011 to September-October 2012. In total, 3520 fish [3380 mollies (Poecilia latipinna) and 140 swordtails (Xiphophorus helleri)] were collected from 10 fish maintenance and breeding centers in Kerman, Iran., Results: Of 3520 fish, only 186 fish (5.3%) were infected with L. cyprinacea, and the remaining fish (94.7%) were not infected. The swordtails (X. helleri) and mollies (P. latipinna) showed the highest (10.7%) and the lowest (5.1%) level of infection, respectively. In other words, there was a significant correlation between species of fish and L. cyprinacea infection (p<0.05). The highest prevalence (39.4%) and the lowest prevalence (0%) of L. cyprinacea were observed during summer and winter, respectively, which can be attributed to the temperature difference between the two seasons. Also, the histopathologic examination of sections revealed some lesions in the epidermis, dermis, and muscles., Conclusion: Considering the existing L. cyprinacea infection in ornamental fish reproduction and breeding centers in Kerman, public knowledge should increase through management methods; physical and chemical treatments should also be applied to inform the public regarding the risk of infection and other internal diseases that may be associated with ornamental fish.

Malaria infection starts with injection of Plasmodium sporozoites by an Anopheles mosquito into the skin of the mammalian host. How sporozoites locate and enter a blood vessel is a critical, but poorly understood process. In this study, we examine sporozoite motility and their interaction with dermal blood vessels, using intravital microscopy in mice. Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained. We find that curvature of sporozoite tracks engaging with vasculature optimizes contact with dermal capillaries. Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

Cysticercosis, especially neurocysticercosis, is a major public health problem in India. We report an unusual case of disseminated cysticercosis with extensive infiltration of the skin, central nervous system, skeletal muscles, eye, lung, and heart. A patient with extensive cutaneous cysticercosis must be thoroughly investigated for widespread internal organ involvement.

The purpose of this study was to characterize the immunopathological response in the skin of S. apella infected with Leishmania (L.) amazonensis and L. (V.) braziliensis parasites, the main causative agents of localized cutaneous leishmaniasis in South America. In infected animals, amastigote forms of L. (L.) amazonensis could be detected till 120 days postinfection (PI), while, in L. (V.) braziliensis infection, parasites could be detected until 180 days PI in the skin sections. CD20(+) cells were detected throughout the experimental time in both groups as well as in CD3(+) cells, which appeared to be activated because high densities of inducible nitric oxide synthase (iNOS(+)) cells were detected at 60 and 90 days PI in both studied groups. After 60 and 120 days PI, decrease in iNOS(+) cells was observed in L. (L.) amazonensis and L. (V.) braziliensis, respectively, which was associated with parasite clearance. Increase in lysozyme(+) cells was observed during the experimental infections, which also can be associated with parasite killing.

The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-β and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-β expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Post-kala-azar dermal leishmaniasis (PKDL) is an uncommon complication of visceral leishmaniasis (VL) but is emerging as an increasingly frequent and serious complication of acquired immunodeficiency syndrome (AIDS). It manifests as a macular, morbilliform, or nodular eruption in a patient who has recovered from VL., Methods: We present two cases of PKDL in the setting of AIDS., Results: These two cases are notable because they demonstrate the severe course of VL, the rare appearance of PKDL in the U.S.A., and the recurrence of disease after >2 years of remission in the second case. Options for treatment include amphotericin B and pentavalent antimonials along with first-line antiretroviral therapy., Conclusions: The prevention of VL relapses, and the development of PKDL in HIV patients with Leishmania co-infection remains challenging. The vast differential diagnosis of other HIV-related cutaneous conditions, along with a lack of experience with this disease, often delays diagnosis. Therapeutic dilemmas concerning drug selection, dosage, scheduling regimen, and the respective durations of initial and maintenance therapy for PKDL need to be addressed. Treatment should aim to target the latent infection and prevent posttreatment VL relapses that may present in an unpredictable fashion. Leishmaniasis remains a recognized but uncommon opportunistic disease in the setting of HIV, which can be associated with atypical features including the appearance of skin lesions years after the treatment of VL., (© 2013 The International Society of Dermatology.)

Interleukin 4 (IL-4) plays a central role in the orchestration of Type 2 immunity. During T cell activation in the lymph node, IL-4 promotes Th2 differentiation and inhibits Th1 generation. In the inflamed tissue, IL-4 signals promote innate and adaptive Type-2 immune recruitment and effector function, positively amplifying the local Th2 response. In this study, we identify an additional negative regulatory role for IL-4 in limiting the recruitment of Th1 cells to inflamed tissues. To test IL-4 effects on inflammation subsequent to Th2 differentiation, we transiently blocked IL-4 during ongoing dermal inflammation (using anti-IL-4 mAb) and analyzed changes in gene expression. Neutralization of IL-4 led to the upregulation of a number of genes linked to Th1 trafficking, including CXCR3 chemokines, CCL5 and CCR5 and an associated increase in IFNγ, Tbet and TNFα genes. These gene expression changes correlated with increased numbers of IFNγ-producing CD4+ T cells in the inflamed dermis. Moreover, using an adoptive transfer approach to directly test the role of IL-4 in T cell trafficking to the inflamed tissues, we found IL-4 neutralization led to an early increase in Th1 cell recruitment to the inflamed dermis. These data support a model whereby IL-4 dampens Th1-chemokines at the site of inflammation limiting Th1 recruitment. To determine biological significance, we infected mice with Leishmania major, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established L. major infection led to a significant increase in the number of IFNγ-producing CD4+ T cells in the infected ear dermis, with no change in the draining LN. Increased lymphocyte influx into the infected tissue correlated with a significant decrease in parasite number. Thus, independent of IL-4's role in the generation of immune effectors, IL-4 attenuates lymphocyte recruitment to the inflamed/infected dermis and limits pathogen clearance.

Infection by malaria parasites begins with the inoculation of sporozoites into the skin of the host. The early events following sporozoite deposition in the dermis are critical for both the establishment of malaria infection and for the induction of protective immune responses. The initial sporozoite inoculum is generally low, and only a small percentage of these sporozoites successfully reach the liver and grow to the next life cycle stage, making this a significant bottleneck for the parasite. Recent studies highlight the importance of sporozoite motility and host cell traversal in dermal exit. Importantly, protective immune responses against sporozoites and liver stages of Plasmodium are induced by dendritic cells in the lymph node draining the skin inoculation site. The cellular, molecular, and immunological events that occur in the skin and associated lymph nodes are the topic of this review.

Effector T cells are critical for clearance of pathogens from sites of infection. Like cytotoxic CD8(+) T cells, CD4(+) helper T cells have been shown to deliver effector molecules directionally toward the immunological synapse, suggesting that infected cells need to be engaged individually to receive effector signals. In contrast, we show here that CD4(+) T cells stably contacted a minority of infected cells, yet these interactions triggered intracellular defense mechanisms in bystander cells in vivo. By using a functional read-out, we provide evidence that this effector bystander activity extends via a gradient of IFN-γ more than 80 μm beyond the site of antigen presentation, promoting pathogen clearance in the absence of immunological synapse formation. Our results thus demonstrate that CD4(+) T cells can exert their protective activity by engaging a minority of infected cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Infection of the mammalian host by schistosome larvae occurs via the skin, although nothing is known about the development of immune responses to multiple exposures of schistosome larvae, and/or their excretory/secretory (E/S) products. Here, we show that multiple (4x) exposures, prior to the onset of egg laying by adult worms, modulate the skin immune response and induce CD4(+) cell hypo-responsiveness in the draining lymph node, and even modulate the formation of hepatic egg-induced granulomas. Compared to mice exposed to a single infection (1x), dermal cells from multiply infected mice (4x), were less able to support lymph node cell proliferation. Analysis of dermal cells showed that the most abundant in 4x mice were eosinophils (F4/80(+)MHC-II(-)), but they did not impact the ability of antigen presenting cells (APC) to support lymphocyte proliferation to parasite antigen in vitro. However, two other cell populations from the dermal site of infection appear to have a critical role. The first comprises arginase-1(+), Ym-1(+) alternatively activated macrophage-like cells, and the second are functionally compromised MHC-II(hi) cells. Through the administration of exogenous IL-12 to multiply infected mice, we show that these suppressive myeloid cell phenotypes form as a consequence of events in the skin, most notably an enrichment of IL-4 and IL-13, likely resulting from an influx of RELMα-expressing eosinophils. We further illustrate that the development of these suppressive dermal cells is dependent upon IL-4Rα signalling. The development of immune hypo-responsiveness to schistosome larvae and their effect on the subsequent response to the immunopathogenic egg is important in appreciating how immune responses to helminth infections are modulated by repeated exposure to the infective early stages of development.

The first step of Plasmodium development in vertebrates is the transformation of the sporozoite, the parasite stage injected by the mosquito in the skin, into merozoites, the stage that invades erythrocytes and initiates the disease. The current view is that, in mammals, this stage conversion occurs only inside hepatocytes. Here, we document the transformation of sporozoites of rodent-infecting Plasmodium into merozoites in the skin of mice. After mosquito bite, ∼50% of the parasites remain in the skin, and at 24 h ∼10% are developing in the epidermis and the dermis, as well as in the immunoprivileged hair follicles where they can survive for weeks. The parasite developmental pathway in skin cells, although frequently abortive, leads to the generation of merozoites that are infective to erythrocytes and are released via merosomes, as typically observed in the liver. Therefore, during malaria in rodents, the skin is not just the route to the liver but is also the final destination for many inoculated parasites, where they can differentiate into merozoites and possibly persist.

Tegumentary leishmaniasis is an important public health problem in several countries. The capacity of the Leishmania species, at the initial moments of the infection, to invade and survive inside the host cells involves the interaction of surface molecules that are crucial in determining the evolution of the disease. Using C57BL/6 wild-type and TLR-2(-/-) mice infected with L. (L.) amazonensis, we demonstrated that TLR-2(-/-) mice presented eosinophilic granuloma in the ear dermis, different from C57BL/6 wild-type mice that presented a cellular profile characterized mainly by mononuclear cell infiltrates, besides neutrophils and eosinophils, during the two first week of infection. When the parasite load was evaluated, we found that the absence of TLR-2 lead to a significant reduction of the infection in deficient mice, when compared with C57BL/6 mice which were more susceptible to the infection. Using TLR-2 deficient mice, it was possible to show that the absence of this receptor determined the reduction of the parasite load and the recruitment of inflammatory cells during the two first weeks after L. (L.) amazonensis infection., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Leishmania (Viannia) shawi was recently characterized and few studies concerning modifications in cellular and humoral immune responses in experimental leishmaniasis have been conducted. In this work, immunopathological changes induced by L. shawi in chronically infected BALB/c mice were investigated. Infected BALB/c mice developed increased lesion size associated with strong inflammatory infiltrate diffusely distributed in the dermis, with highly infected macrophages. The humoral immune response was predominantly directed toward the IgG1 isotype. The functional activity of CD4(+) and CD8(+) T cells showed significantly increased TNF-alpha mRNA levels associated with reduced IFN-gamma expression by CD4(+) T cells and the double negative (dn) CD4CD8 cell subset. High IL-4 levels expressed by CD8(+) T cells and dnCD4CD8 and TGF-beta by CD4(+) and CD8(+) T cells were detected, while IL-10 was highly expressed by all three cell subpopulations. Taken together, these results show an evident imbalance between TNF-alpha and IFN-gamma that is unfavorable to amastigote replication control. Furthermore, L. shawi seems to regulate different cell populations to express deactivating cytokines to avoid its own destruction. This study indicates BALB/c mice as a potentially good experimental model for further studies on American cutaneous leishmaniosis caused by L. shawi.

Regulatory T cells (Tregs) play a central role in maintenance of immune homeostasis by controlling harmful immune responses to inappropriate antigens and are thought to play a key role in modulating hypersensitivity reactions. Infestation of sheep with Psoroptes ovis results in a pronounced cutaneous hypersensitivity-type response, which appears to be crucial for mite survival. We hypothesize that (i) Tregs are involved in sheep scab lesions and (ii) Treg responses may crucially affect lesion development and subsequent mite survival. Foxp3 is a key transcription factor required for generation and maintenance of Tregs in rodents and humans, and is the most widely used marker for Tregs in these species. In this study, we sequence ovine foxp3 and show that it exhibits a high degree of homology with foxp3 from other species. Using a validated immunohistochemical staining technique, we demonstrate that infestation of sheep with P. ovis results in an influx of Foxp3(+) T cells into the skin. Future work will investigate the regulatory function of ovine Foxp3(+) T cells and determine whether the quality of the Treg response to P. ovis plays a role in individual susceptibility to the mite.

Indian post-kala-azar dermal leishmaniasis (PKDL) is a low-frequency (5-10%) dermal sequela of visceral leishmaniasis (VL) caused by Leishmania donovani; importantly, affected individuals are speculated to be parasite reservoirs. Insight into its immunopathogenesis could translate into rational immunomodulatory therapeutic approaches against leishmaniases. In patients with PKDL (n=21), peripheral lymphocytes were analyzed for surface markers, intracellular cytokines, and lymphoproliferative responses using flow cytometry. In lesional tissue biopsies (n=12), expression of counter-regulatory cytokines (IFN-gamma and IL-10) and the T-regulatory transcription factor forkhead box protein 3 (Foxp3) was analyzed using reverse transcriptase-PCR, along with immunohistochemical detection (n=8) of CD3 and Foxp3 positivity. In patients with PKDL, circulating CD8(+)CD28(-) and antigen-induced IL-10(+)CD3(+) lymphocytes were increased and receded with treatment. CD8(+) lymphocytes showed impaired proliferative responses to L. donovani antigen (LDA) and phytohemagglutinin, which were reinstated after treatment. At presentation, the upregulated lesional IFN-gamma and IL-10 messenger RNA (mRNA), Foxp3 mRNA, and protein were curtailed after treatment. In Indian patients with PKDL, increased frequency of the CD8(+)CD28(-) phenotype, enhanced antigen-specific IL-10 production, and accompanying anergy of circulating lymphocytes suggest their regulatory nature. Furthermore, the concomitantly elevated lesional expression of Foxp3 suggests their possible recruitment into the lesional site, which would sustain disease pathology.

Topical therapy is an attractive approach for the treatment of Leishmania major cutaneous leishmaniasis (CL). WR279396, an expanded-spectrum aminoglycoside ointment, is now in phase 3 trials. Because the application of a cream is easier than the injection of pentavalent antimony, many patients with CL will likely be treated with WR279396 soon after the onset of a lesion. However, this new therapeutic approach may impair the acquisition of immunity. We evaluated the impact of early topical therapy on acquired immunity in an optimized mouse model of L. major-induced CL. The efficacy of the WR279396 ointment in this model has been established previously. Acquired immunity was defined as the absence of lesions upon reinoculation of the same parasite isolate at a different skin site. Bioluminescence-based follow-up of luciferase-expressing L. major loads was also performed. In this model, the control of L. major loads at the initial inoculation site and the acquisition of immunity are simultaneous (day 22 postinoculation). The clinical and parasitological efficacies of WR279396 applied as early as day 11 postinoculation, i.e., during the L. major multiplication phase, did not impair the acquisition of immunity to a second L. major challenge. This is reassuring from the perspective of the wide deployment of WR279396-based therapy in foci where L. major is endemic.

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is a parasitic disease leading to debilitating skin disease and blindness, with major economic and social consequences. The pathology of onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses. In onchocerciasis a subcutaneous nodule is formed around the female worms, the core of which is a dense infiltrate of inflammatory cells in which microfilariae are released. It has been established that the formation of nodules is associated with angiogenesis. In this study, we show using specific markers of endothelium (CD31) and lymphatic endothelial cells (Lyve-1, Podoplanin) that not only angiogenesis but also lymphangiogenesis occurs within the nodule. 7% of the microfilariae could be found within the lymphatics, but none within blood vessels in these nodules, suggesting a possible route of migration for the larvae. The neovascularisation was associated with a particular pattern of angio/lymphangiogenic factors in nodules of onchocerciasis patients, characterized by the expression of CXCL12, CXCR4, VEGF-C, Angiopoietin-1 and Angiopoietin-2. Interestingly, a proportion of macrophages were found to be positive for Lyve-1 and some were integrated into the endothelium of the lymphatic vessels, revealing their plasticity in the nodular micro-environment. These results indicate that lymphatic as well as blood vascularization is induced around O. volvulus worms, either by the parasite itself, e.g. by the release of angiogenic and lymphangiogenic factors, or by consecutive host immune responses.

The original description of Henneguya pellis, a myxozoan parasitizing blue catfish Ictalurus furcatus, is supplemented with new data on histopathology, spore morphology, and 18S small subunit (SSU) ribosomal DNA (rDNA) sequence. Plasmodia presented as both internal and external, raised, cyst-like lesions on the body wall of the peritoneal cavity and on the skin. The cysts contained numerous elongate, lanceolate myxospores, flattened parallel to the suture line. The spore body was 14.8 ± 1.1 µm (range 13.0-17.1) long and 4.8 ± 0.8 µm (range 4.0-7.4) wide in frontal view. The caudal appendages were 77.7 ± 8.8 (range 57.4-96.4) in length. There were 2 pyriform polar capsules, unequal in length, with the longer capsule measuring 7.2 ± 0.6 µm (range 6.2-8.4) in length and the shorter capsule measuring 6.5 ± 0.5 µm (range 5.5-8.0). The polar capsules were not significantly different in width, measuring 1.7 ± 0.2 µm (range 1.4-1.9). There were 8 turns in the polar filament coil. The total length of the spore was 92.5 ± 9.2 µm (range 73.3-113.5). Spore morphology and site of development are similar to that of Henneguya sutherlandi from channel catfish; however, 18S rDNA sequence data support previous findings that identify H. pellis and H. sutherlandi as 2 distinct species.

Sporozoites are the infective stage of the malaria parasite. They are deposited in the skin by infected Anopheles mosquitoes and must penetrate cell barriers in the skin and liver sinusoid to reach their target cell, the hepatocyte, where they enter in a vacuole and begin development into the next life cycle stage, the exoerythrocytic form. Recent advances in our understanding of sporozoite biology in the dermal inoculation site, the role of cell traversal and the mechanism by which sporozoites productively invade hepatocytes will be highlighted in this review.

Background: Sand fly saliva has an array of pharmacological and immunomodulatory components, and immunity to saliva protects against Leishmania infection. In the present study, we have studied the immune response against Lutzomyia intermedia saliva, the main vector of Leishmania braziliensis in Brazil, and the effects of saliva pre-exposure on L. braziliensis infection employing an intradermal experimental model., Methodology/principal Findings: BALB/c mice immunized with L. intermedia salivary gland sonicate (SGS) developed a saliva-specific antibody response and a cellular immune response with presence of both IFN-gamma and IL-4. The inflammatory infiltrate observed in SGS-immunized mice was comprised of numerous polymorphonuclear and few mononuclear cells. Mice challenged with live L. braziliensis in the presence of saliva were not protected although lesion development was delayed. The inoculation site and draining lymph node showed continuous parasite replication and low IFN-gamma to IL-4 ratio, indicating that pre-exposure to L. intermedia saliva leads to modulation of the immune response. Furthermore, in an endemic area of cutaneous leishmaniasis, patients with active lesions displayed higher levels of anti-L. intermedia saliva antibodies when compared to individuals with a positive skin test result for Leishmania., Conclusion: These results show that pre-exposure to sand fly saliva plays an important role in the outcome of cutaneous leishmaniasis, in both mice and humans. They emphasize possible hurdles in the development of vaccines based on sand fly saliva and the need to identify and select the individual salivary candidates instead of using whole salivary mixture that may favor a non-protective response.

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. The steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo.

Malaria, the disease caused by Plasmodium, kills more than 1 million people annually. Little is known of the pre-erythrocytic phase of the parasite life cycle, i.e., after the sporozoite stage is inoculated in the dermis by a mosquito and before the erythrocyte-infecting stage is released from hepatocytes. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We describe previously unrecognized steps in the parasite's journey to the liver of the host, which are likely to play an important role in the host immune response.

Despite being central to parasite establishment and subsequent host pathological and immunologic responses, host-parasite interactions during early third-stage filarial larva (L3) migration are poorly understood. These studies aimed to define early tissue migration of Brugia pahangi L3 in the gerbil (Meriones unguiculatus) and measure host cellular responses during this period. Gerbils were intradermally inoculated in the hind limb with 100 B. pahangi L3, and necropsies were performed at various times. At 3 h, most L3 (96.3%) were recovered from tissues associated with the infection site, with marked L3 migration occurring by 24 h. Larvae were dispersed throughout the lymphatics at 7 days postinfection (dpi), and at 28 dpi, most parasites were recovered from the spermatic cord lymphatics. Parasites were identified histologically at all time points. Inflammatory cells, primarily neutrophils, were frequently observed around larvae in the dermis and muscle near the injection site at 3 h and 24 h. Levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha mRNA peaked at 3 h in all tissues, with IL-6 levels also high in the spleen at 28 dpi. Levels of IL-4 mRNA were elevated in all tissues at 28 dpi. These observations demonstrate that L3 migrate quickly through various tissues and into lymph nodes in a predictable pattern. Migrating L3 induce an early acute inflammatory response that is modulated as parasites establish in the lymphatics. Polarization of the host response towards a dominant Th2-like profile is present at 7 dpi and is well established by 28 dpi in this permissive host.

The diagnosis of cutaneous leishmaniasis (CL) may depend on the detection of the parasite in histologic sections, the growth of the promastigotes in culture, or the identification of parasite by other techniques. We performed polymerase chain reaction (PCR) on paraffin-embedded biopsies to determine the validity of this technique for diagnosis of CL. PCR was used to detect the parasite using 2 different DNA extraction methods. PCR was positive in all 20 cases when the Leishmania parasite was detected by light microscopy. Twenty-seven of 34 cases that were negative microscopically for the parasite were positive using PCR. The first extraction method of DNA identified leishmanial DNA in 41 of 54 cases (75.9%); the second extraction of DNA was positive in 47 of 54 cases (87%). PCR was negative in all of the nonleishmaniasis cases. The PCR-based method appears to be a useful diagnostic approach for identification of suspected cases of CL.

Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis (ACL) and mucocutaneous leishmaniasis. In the present study, we have developed an experimental model of infection that closely resembles ACL caused by L. braziliensis. In order to do so, BALB/c mice were infected in the ear dermis with 10(5) parasites and distinct aspects of the infection were evaluated. Following inoculation, parasite expansion in the ear dermis was accompanied by the development of an ulcerated dermal lesion which healed spontaneously, as seen by the presence of a scar. Histological analysis of infected ears showed the presence of a mixed inflammatory infiltrate consisting of both mononuclear and polymorphonuclear cells. In draining lymph nodes, parasite replication was detected throughout the infection. In vitro restimulation of draining lymph node cells followed by intracellular staining showed an up-regulation in the production of gamma interferon (IFN-gamma) and in the frequency of IFN-gamma-secreting CD4(+) and CD8(+) T cells. Reverse transcription-PCR of ears and draining lymph node cells showed the expression of CC chemokines. The dermal model of infection with L. braziliensis herein is able to reproduce aspects of the natural infection, such as the presence of an ulcerated lesion, parasite dissemination to lymphoid areas, and the development of a Th1-type immune response. These results indicate that this model shall be useful to address questions related to the concomitant immunity to reinfection and parasite persistence leading to mucocutaneous leishmaniasis.

The migration of Schistosoma japonicum and S. mansoni through mouse skin epidermis and dermis was compared by immunofluorescence techniques from 4 to 22 h after infection. At all times, the percentage of parasites detected in the dermis was significantly higher for S. japonicum than for S. mansoni. Thus, S. japonicum migrates more rapidly very early after infection. This agrees with the quicker migration observed previously by this species for later times. Both species expressed antigens related to the cercarial glycocalyx on the parasite body and antigenically detectable elastase in the acetabular glands, at least until 22 h after infection. Bot sets of antigens were also left as "traces" in cercarial migration channels in the skin as well as in skin tissue in the absence of detectable worms or migration channels. The data further substantiate differences between schistosome species in the speed of migration, and suggest that glycocalyx-related antigens and cercarial elastase continue to be expressed for at least 1 day after infection.

Human leishmaniasis is caused by protozoan Leishmania (L.) parasites and comprises a heterogeneous group of clinical appearances ranging from visceral to cutaneous leishmaniasis. In the New World, L. mexicana mediates American cutaneous leishmaniasis, one of the most common forms of this disease. Two different disease progressions can be observed: (i) self-healing localized cutaneous leishmaniasis (LCL) and (ii) progressive diffuse cutaneous leishmaniasis (DCL). These different forms are associated with a T helper 1 (Th1) or Th2 response, respectively, and are additionally characterized by opposing dermal chemokine profiles. Lesions of LCL show high expression of CCL2/MCP-1, CXCL9/MIG, CXCL10/IP-10 and only low amounts of CCL3/MIP-1alpha. In contrast, lesions of chronic DCL are dominated by the expression of CCL3/MIP-1alpha. This finding implies that CCL2/MCP-1 contributes to the healing process. Indeed, CCL2/MCP-1 induces leishmanicidal activities in human monocytes in contrast to CCL3/MIP-1alpha. This effect is enhanced by interferon-gamma and abrogated by interleukin-4. In the murine model of leishmaniasis, the impact of CCL2/MCP-1 is well documented. Normally resistant mice become susceptible for Leishmania infections if CCR2, the receptor for CCL2/MCP-1, is knocked out. Based on this evidence, we propose that tissue specific expression of these small molecules actively regulates cell traffic and tissue localization of effector cells and, additionally, has direct immunological effects.

Despite the fact that Leishmania are transmitted exclusively by sand flies, none of the experimental models of leishmaniasis have established infection via sand fly bites. Here we describe a reproducible murine model of Leishmania major infection transmitted by Phlebotomus papatasi. Prior exposure of mice to bites of uninfected sand flies conferred powerful protection against Leishmania major that was associated with a strong delayed-type hypersensitivity response and with interferon-gamma production at the site of parasite delivery. These results have important implications for the epidemiology of cutaneous leishmaniasis and suggest a vaccination strategy against this and possibly other vector-borne diseases.

About ten cases of filariosis have recently been recorded in the Hungarian medical literature, six of them caused by Dirofilaria repens. Dirofilaria repensis a mosquito-transmitted filaroid worm in the subcutaneous tissue of dogs and cats in the temperate areas of the Old World. It accidentally infects man, too, and can remain unidentified due to physicians, poor knowledge of the parasite. In the last two years six new Dirofilaria repensinfections have been found in various parts of the country: five localised dermally and one in the deep tissues. Two of the cases might have been acquired in Italy during summer travels. Four patients, however, have never been abroad, these cases must be considered autochtonous infections. The thickness of the multilayered cuticle of the worm, diameter of the body and the size, form and number of the longitudinal ridges on its surface are used in the histological diagnosis of the the parasite.

We have developed a model of cutaneous leishmaniasis due to Leishmania major that seeks to mimic the natural conditions of infection. 1,000 metacyclic promastigotes were coinoculated with a salivary gland sonicate (SGS) obtained from a natural vector, Phlebotomus papatasii, into the ear dermis of naive mice or of mice preexposed to SGS. The studies reveal a dramatic exacerbating effect of SGS on lesion development in the dermal site, and a complete abrogation of this effect in mice preexposed to salivary components. In both BALB/c and C57Bl/6 (B/6) mice, the dermal lesions appeared earlier, were more destructive, and contained greater numbers of parasites after infection in the presence of SGS. Furthermore, coinoculation of SGS converted B/6 mice into a nonhealing phenotype. No effect of SGS was seen in either IL-4- deficient or in SCID mice. Disease exacerbation in both BALB/c and B/6 mice was associated with an early (6 h) increase in the frequency of epidermal cells producing type 2 cytokines. SGS did not elicit type 2 cytokines in the epidermis of mice previously injected with SGS. These mice made antisaliva antibodies that were able to neutralize the ability of SGS to enhance infection and to elicit IL-4 and IL-5 responses in the epidermis. These results are the first to suggest that for individuals at risk of vector-borne infections, history of exposure to vector saliva might influence the outcome of exposure to transmitted parasites.