Recent evolution of extreme cestode growth suppression by a vertebrate host.

Parasites can be a major cause of natural selection on hosts, which consequently evolve a variety of strategies to avoid, eliminate, or tolerate infection. When ecologically similar host populations present disparate infection loads, this natural variation can reveal immunological strategies underlying adaptation to infection and population divergence. For instance, the tapeworm Schistocephalus solidus persistently infects 0-80% of threespine stickleback (Gasterosteus aculeatus) in lakes on Vancouver Island. To test whether these heterogeneous infection rates result from evolved differences in immunity, we experimentally exposed laboratory-reared fish from ecologically similar high-infection and no-infection populations to controlled doses of Schistocephalus We observed heritable between-population differences in several immune traits: Fish from the naturally uninfected population initiated a stronger granulocyte response to Schistocephalus infection, and their granulocytes constitutively generate threefold more reactive oxygen species in cell culture. Despite these immunological differences, Schistocephalus was equally successful at establishing initial infections in both host populations. However, the no-infection fish dramatically suppressed tapeworm growth relative to high-infection fish, and parasite size was intermediate in F1 hybrid hosts. Our results show that stickleback recently evolved heritable variation in their capacity to suppress helminth growth by two orders of magnitude. Data from many natural populations indicate that growth suppression is widespread but not universal and, when present, is associated with reduced infection prevalence. Host suppression of helminth somatic growth may be an important immune strategy that aids in parasite clearance or in mitigating the fitness costs of persistent infection.

Hepatozoon ellisgreineri n. sp. (Hepatozoidae): description of the first avian apicomplexan blood parasite inhabiting granulocytes.

Blood parasites of the genus Hepatozoon (Apicomplexa, Hepatozoidae) infect all groups of terrestrial vertebrates, and particularly high prevalence and species diversity have been reported in reptiles and mammals. A few morphologically similar species, in which gamonts inhabit mononuclear leukocytes and red blood cells, have been described in birds. Here, we report a new Hepatozoon species, which was found in wild-caught secretary birds Sagittarius serpentarius, from Tanzania. Hepatozoon ellisgreineri n. sp. can be readily distinguished from all described species of avian Hepatozoon because its gamonts develop only in granulocytes, predominantly in heterophils, a unique characteristic among bird parasites of this genus. Additionally, this is the first reported avian apicomplexan blood parasite, which inhabits and matures in granulocytes. We describe H. ellisgreineri based on morphological characteristics of blood stages and their host cells. This finding broadens knowledge about host cells of avian Hepatozoon spp. and other avian apicomplexan blood parasites, contributing to the better understanding of the diversity of haematozoa. This is the first report of hepatozoonosis in endangered African birds of the Sagittariidae.

Low-level regulatory T-cell activity is essential for functional type-2 effector immunity to expel gastrointestinal helminths.

Helminth infection is frequently associated with the expansion of regulatory T cells (Tregs) and suppression of immune responses to bystander antigens. We show that infection of mice with the chronic gastrointestinal helminth Heligmosomoides polygyrus drives rapid polyclonal expansion of Foxp3(+)Helios(+)CD4(+) thymic (t)Tregs in the lamina propria and mesenteric lymph nodes while Foxp3(+)Helios(-)CD4(+) peripheral (p)Treg expand more slowly. Notably, in partially resistant BALB/c mice parasite survival positively correlates with Foxp3(+)Helios(+)CD4(+) tTreg numbers. Boosting of Foxp3(+)Helios(+)CD4(+) tTreg populations by administration of recombinant interleukin-2 (rIL-2):anti-IL-2 (IL-2C) complex increased worm persistence by diminishing type-2 responsiveness in vivo, including suppression of alternatively activated macrophage and granulomatous responses at the sites of infection. IL-2C also increased innate lymphoid cell (ILC) numbers, indicating that Treg functions dominate over ILC effects in this setting. Surprisingly, complete removal of Tregs in transgenic Foxp3-DTR mice also resulted in increased worm burdens, with "immunological chaos" evident in high levels of the pro-inflammatory cytokines IL-6 and interferon-γ. In contrast, worm clearance could be induced by anti-CD25 antibody-mediated partial depletion of early Treg, alongside increased T helper type 2 responses and without incurring pathology. These findings highlight the overarching importance of the early Treg response to infection and the non-linear association between inflammation and the prevailing Treg frequency.

Lipocalin 2 bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism.

Plasmodium parasites multiply within host erythrocytes, which contain high levels of iron, and parasite egress from these cells results in iron release and host anemia. Although Plasmodium requires host iron for replication, how host iron homeostasis and responses to these fluxes affect Plasmodium infection are incompletely understood. We determined that Lipocalin 2 (Lcn2), a host protein that sequesters iron, is abundantly secreted during human (P. vivax) and mouse (P. yoeliiNL) blood-stage malaria infections and is essential to control P. yoeliiNL parasitemia, anemia, and host survival. During infection, Lcn2 bolsters both host macrophage function and granulocyte recruitment and limits reticulocytosis, or the expansion of immature erythrocytes, which are the preferred target cell of P. yoeliiNL. Additionally, a chronic iron imbalance due to Lcn2 deficiency results in impaired adaptive immune responses against Plasmodium parasites. Thus, Lcn2 exerts antiparasitic effects by maintaining iron homeostasis and promoting innate and adaptive immune responses.

Cutting edge: mast cells critically augment myeloid-derived suppressor cell activity.

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (Kit(Wsh)(/)(Wsh)) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Granulocytes in helminth infection -- who is calling the shots?

Helminths are parasitic organisms that can be broadly described as "worms" due to their elongated body plan, but which otherwise differ in shape, development, migratory routes and the predilection site of the adults and larvae. They are divided into three major groups: trematodes (flukes), which are leaf-shaped, hermaphroditic (except for blood flukes) flatworms with oral and ventral suckers; cestodes (tapeworms), which are segmented, hermaphroditic flatworms that inhabit the intestinal lumen; and nematodes (roundworms), which are dioecious, cylindrical parasites that inhabit intestinal and peripheral tissue sites. Helminths exhibit a sublime co-evolution with the host's immune system that has enabled them to successfully colonize almost all multicellular species present in every geographical environment, including over two billion humans. In the face of this challenge, the host immune system has evolved to strike a delicate balance between attempts to neutralize the infectious assault versus limitation of damage to host tissues. Among the most important cell types during helminthic invasion are granulocytes: eosinophils, neutrophils and basophils. Depending on the specific context, these leukocytes may have pivotal roles in host protection, immunopathology, or facilitation of helminth establishment. This review provides an overview of the function of granulocytes in helminthic infections.

Endothelial nitric oxide synthase limits the inflammatory response in mouse cutaneous leishmaniasis.

Endothelial nitric oxide synthase (eNOS) was originally discovered in the cardiovascular system, where it contributes to the regulation of blood pressure and the inhibition of platelet adhesion. Considering that the vascular endothelium is critical for the initiation of inflammatory processes and that eNOS has been detected in certain types of immune cells, we investigated the function of eNOS in C57BL/6 mice infected with Leishmania major, a protozoan parasite that causes a chronic, but self-healing skin disease. C57BL/6 eNOS(-/-) mice developed more severe (but ultimately resolving) skin lesions with strikingly higher numbers of parasites compared to wildtype controls. In accordance with our finding that naive T lymphocytes and Th1 cells (as well as Th2 cells) did not express eNOS after stimulation and that eNOS was not required for Th1 differentiation in vitro, lymph node T cells from L. major-infected wildtype and eNOS(-/-) mice released comparable amounts of IFN-gamma and proliferated equally well. Immunohistological analyses revealed that the expression of inducible NO synthase in the skin and draining lymph nodes of infected mice was completely preserved in the absence of eNOS. However, the skin lesions of eNOS(-/-) mice were characterized by massive infiltrates of granulocytes, which in vitro similar to inflammatory macrophages failed to express eNOS. From these data, we conclude that during cutaneous leishmaniasis eNOS-derived NO, presumably released by vascular endothelial cells, counteracts the recruitment of granulocytes, which are known to function as host cells and trojan horses for Leishmania parasites, and thereby limits the severity of the skin lesions.

Evaluation of endocytic capacity and NADPH-oxidase activity from armadillo (Dasypus novemcinctus) eosinophils infected with microfilariae.

Endocytic activity of phagocytic cells from armadillos infected with viruses, parasites or bacteria is unknown. This report shows that eosinophils from armadillos infected with microfilaria act against these helmintic parasites but have deficiencies in their oxygen-dependent bacteriocidal mechanisms and also in endocytic capacity against yeast.

Uninfected erythrocytes inhibit Plasmodium falciparum-induced cellular immune responses in whole-blood assays.

Whole-blood assays (WBAs) have been successfully used as a simple tool for immuno-epidemiological field studies evaluating cellular immune responses to mycobacterial and viral antigens. Rather unexpectedly, we found very poor cytokine responses to malaria antigens in WBAs in 2 immuno-epidemiological studies carried out in malaria endemic populations in Africa. We have therefore conducted a detailed comparison of cellular immune responses to live (intact) and lysed malaria-infected erythrocytes in WBAs and in peripheral blood mononuclear cell (PBMC) cultures. We observed profound inhibition of both proliferative and interferon-gamma responses to malarial antigens in WBAs as compared with PBMC cultures. This inhibition was seen only for malaria antigens and could not be overcome by increasing either antigen concentration or responder cell numbers. Inhibition was mediated by intact erythrocytes and occurred early in the culture period, suggesting that failure of antigen uptake might underlie the lack of T-cell responses. In support of this hypothesis, we have shown that intact uninfected erythrocytes specifically inhibit phagocytosis of infected red blood cells by peripheral blood monocytes. We propose that specific biochemical interactions with uninfected erythrocytes inhibit the phagocytosis of malaria-infected erythrocytes and that this may impede T-cell recognition in vivo.

Silica treatment increases the susceptibility of the Cabo Frio strain of Biomphalaria tenagophila to Schistosoma mansoni infection but does not alter the natural resistance of the Taim strain.

The present work demonstrates that silica treatment represents a suitable in vivo method to evaluate the role of host phagocytic hemocytes in the mechanisms of resistance to parasitic infection. Silica inoculation into Biomphalaria tenagophila snail induced a significant reduction in the circulating hemolymph granulocytes in both strains tested (Taim and Cabo Frio). The granulocyte reduction was accompanied by a significant increase in the number of circulating dead cells. In B. tenagophila Cabo Frio, silica treatment enhanced snail susceptibility to Schistosoma mansoni, shortening the intramolluskan phase of the parasite and increasing the number of sporocysts and cercariae produced. In B. tenagophila Taim, the same treatment did not abrogate natural resistance to S. mansoni reported for this snail strain. These in vivo results demonstrate that macrophage-like granulocytes are involved in the mechanism of S. mansoni sporocyst destruction in Cabo Frio snails and suggest that another, different mechanism may be responsible to the natural resistance of B. tenagophila Taim.

Changes in circulating and tissue-infiltrating hemocyte parameters of European flat oysters, Ostrea edulis, naturally infected with Bonamia ostreae.

We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti.

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.

Spatial analysis of human granulocytic ehrlichiosis near Lyme, Connecticut.

Geographic information systems combined with methods of spatial analysis provide powerful new tools for understanding the epidemiology of diseases and for improving disease prevention and control. In this study, the spatial distribution of a newly recognized tick-borne disease, human granulocytic ehrlichiosis (HGE), was investigated for nonrandom patterns and clusters in an area known to be endemic for tick-borne diseases. Analysis of confirmed cases of HGE identified in 1997-2000 in a 12-town area around Lyme, Connecticut, showed that HGE infections are not distributed randomly. Smoothed HGE incidence was higher around the mouth of the Connecticut River and lower to the north and west. Cluster analysis identified one area of increased HGE risk (relative risk=1.8, p=0.001). This study demonstrates the utility of geographic information systems and spatial analysis to clarify the epidemiology of HGE.

Monoclonal antibodies specific for the two types of wall-forming bodies of Eimeria tenella macrogametes (Coccidia, Apicomplexa).

Two monoclonal antibodies (mAbs) raised against the macrogamonts of Eimeria tenella identified antigens located in the wall-forming bodies of type I (WF I) and type II (WF II) by indirect immunofluorescence and by immunoelectron microscopy. With these mAbs, the involvement of both types of wall-forming body at the protein level in the formation of the inner and outer oocyst walls of E. tenella was shown by indirect immunofluorescence assay. On Western blots of pure macrogamont, mAb E1D8 against WF I reacted with a series of bands between 42 kDa and 105 kDa. In pure, unsporulated extract, this mAb recognized a complex of bands between 26 kDa and 153 kDa. mAb E2E5 against WF II, on Western blots of pure extract of macrogamonts, recognized an antigen of 51 kDa. Later in the development, after the formation of the inner oocyst wall, mAb E2E5 reacted with three polypeptide of 23, 25 and 30 kDa. Proteolytic processing may be forwarded as the mechanism regulating the distinct regulation protein involved in the oocyst wall.

[Trypanosomiasis--a real risk for tourists visiting national parks in Tanzania]. / Trypanosomiasis--reell risiko for turister i Tanzanias nasjonalparker.

African sleeping sickness is no longer a rare disease among tourists visiting national parks in Tanzania. The disease is caused by a parasite, Trypanosoma brucei, which is transmitted by the tsetse fly. Two species infect humans: Trypanosoma brucci gambiense and Trypanosoma brucei rhodesiense; the last form is re-emerging in parts of Africa. Untreated this disease carries a mortality of nearly 100%. This article describes a case of African sleeping sickness in a tourist visiting Tanzania, which was diagnosed at the Nordic Clinic, Dar es Salaam. The most important symptoms, diagnostic investigations as well as the main principles of treatment are described. Patients with this condition need to be admitted and treated at centres with competence in tropical diseases. African sleeping sickness should be kept in mind in tourists returning to their home country with fever after visits to national parks in Eastern Africa. With early treatment, cure is almost certain. The only way to prevent this condition is through protection against bites of the tsetse fly.

Identification and quantification of granulocytes in caecal mucosa and submucosa of chickens experimentally infected with Eimeria tenella and Salmonella enteritidis.

1. Poultry granulocytes are not clearly distinguished from each other with haematoxylin-eosin (HE) stain; thus, histochemical techniques must be used. Three experiments were carried out using 4-week-old Leghorn chickens. 2. Three, 80-chicken groups were orally infected with (1) 10(8) colony forming units (CFUs) Salmonella enteritidis, or (2) 10(4) Eimeria tenella oocysts, or (3) 10(8) CFUs S. enteritidis + 10(4) E. tenella oocysts. Ten chickens from each group were euthanased and caecum samples obtained. Caecum samples were fixed in 10% formalin (buffered, pH 7.4) at 4, 8, 12 h, 1, 3, 5, 7, and 14 d post-inoculation (PI). 3. Samples were stained using three different staining techniques: HE for the identification of heterophils and eosinophils, Ziehl-Neelsen for mast cells, and p-phenilenediamine dihydrochloride plus pyrocatechol (PPD + PC) for eosinophils. 4. Birds from Experiment 1 showed no changes in the numbers of granulocytes. Birds from Experiments 2 and 3 showed higher numbers of heterophils in caecal mucosa and submucosa separately, on d 5 and 7. In Experiment 3, a decrease was observed in submucosal mast cells on d 3. Chickens from Experiments 2 and 3 showed increased numbers of mucosal mast cells between d 7 and 14. 5. PPD + PC positively stained eosinophils, but not heterophils. 6. Numbers of heterophils and mast cells were increased during the acute inflammatory process caused by E. tenella. Therefore, mast cells could play a role as primary inflammatory cells. Eosinophils seem not to be part of the inflammatory process caused by E. tenella.

Human granulocytic ehrlichiosis in pancreas transplant recipients.

Human ehrlichioses are tick-borne infections caused by bacteria in the genus Ehrlichia of the family Rickettsiaceae. To date there have been three cases of ehrlichiosis reported in the transplant population, a human monocytic ehrlichiosis (HME) infection in a liver transplant recipient and two cases of human granulocytic ehrlichiosis (HGE) in kidney transplant recipients. We report three pancreas transplant patients who developed HGE in the last two years at a single southeastern center in the United States. All three patients had clinical, laboratory, and pathophysiologic findings on bone marrow biopsy and peripheral blood smears consistent with HGE, and responded to doxycycline therapy. In the setting of potent immunosuppression, ehrlichiosis should be considered in the differential diagnosis of transplant patients presenting with persistent fever, pancytopenia, and abnormal liver function. Patients with ehrlichiosis infection may be at risk for developing other opportunistic infections or lymphoproliferative disease.

Role of IL-5 in innate and adaptive immunity to larval Strongyloides stercoralis in mice.

Protective immunity to Strongyloides stercoralis infective larvae in mice has been shown to be dependent on IL-5 based on mAb depletion studies. The goal of this study was to determine the functional role of IL-5 during the innate and adaptive immune response to larval S. stercoralis in mice. In these studies, three strains of mice were used: wild-type C57BL/6J (WT), IL-5 knockout (KO), and IL-5 transgenic (TG). Innate responses to the larvae indicated that there was enhanced survival in the KO animals and decreased survival in the TG animals compared with WT. Furthermore, killing of larvae in TG mice was associated with eosinophil infiltration and degranulation. In studying the adaptive immune response, it was observed that immunization of KO mice did not lead to the development of protective immunity. Experiments were then performed to determine whether KO mice reconstituted with Abs or cells could then develop protective immunity. KO mice displayed protective immunity via a granulocyte-dependent mechanism following injection of purified IgM from immune wild-type animals. Immunity in KO mice could also be reconstituted by the injection of eosinophils at the time of immunization. These eosinophils did not participate in actively killing the challenge infection, but rather were responsible for the induction of a protective Ab response. We conclude that IL-5 is required in the protective immune response for the production of eosinophils, and that eosinophils were involved in larval killing during innate immunity and in the induction of protective Abs in the adaptive immune response.

Value of the quantitative buffy coat capillary tube test (QBC) in the microscopic diagnosis of bancroftian filariasis.

The diagnostic performance of the commerical quantitative buffy coat capillary tube technique was compared with the standard diagnosis of filariasis by microscopical examination of Giemsa stained thick blood films. The comparison was conducted among 83 subjects. The QBC test was highly sensitive than the thick blood film in detection of microfilaraemic patients. The percentage of positive cases among 35 subjects with fever and enlarged lymph nodes in the axilla or in the groin was 48.6% versus 42.8% by thick blood film. The sensitivity was 94.4% versus 83.3% in 18 subjects with lymphatic varices or hydrocele. The sensitivity of the QBC capillary tubes was poor (20%) versus 6.6% among 30 filarial patients with different degrees of lymphatic edema. It is concluded that the QBC capillary test is very simple, rapid and convenient as a diagnostic method for microfilaraemic patients but not suggested to be used neither in the quantitation of the parasite numbers or in species identification.

Analysis of immune lesions in neurocysticercosis patients: central nervous system response to helminth appears Th1-like instead of Th2.

Neurocysticercosis (NCC) caused by the helminth Taenia solium is the most common parasitic infection of the human central nervous system (CNS) worldwide. Because clinical symptoms are associated with localized immunological responses in the brain, characterization of these responses are pivotal for understanding the pathogenesis of cysticercosis. Immunohistochemical analysis of brain specimens from several patients with cysticercosis revealed at least four types of immune responses, including: (i) an antibody response (IgM + plasma cells), (ii) a predominant NK response, (iii) an infiltrate with abundant macrophages and granulocytes, and (iv) an intense infiltrate with a predominance of macrophages and T cells. The intensity and type of immunity appeared to be associated somewhat with the parasite's viability and anatomical location. In most of the lesions, cell mediated responses were evident and proinflammatory cytokines including IL12 predominated. Moreover, IL4 was undetectable in the immune infiltrates. Thus, the CNS response to this helminth, unlike the systemic response, is predominately Th1-like.