Evaluation of a new live recombinant vaccine against cutaneous leishmaniasis in BALB/c mice.

BACKGROUND: Leishmaniasis is a serious health problem in some parts of the world. In spite of the many known leishmaniasis control measures, the disease has continued to increase in endemic areas, and no effective vaccine has been discovered. METHODS: In this study, Leishmania tarentulae was used as a living factory for the production of two LACK and KMP11 immunogenic antigens in the mice body, and safety profiles were investigated. The sequences of the KMP11 and LACK L. major antigens were synthesized in the pLEXSY-neo 2.1 plasmid and cloned into E. coli strain Top10, and after being linearized with the SwaI enzyme, they were transfected into the genome of L. tarentolae. The L. tarentolae-LACK/KMP11/EGFP in the stationary phase with CpG ODN as an adjuvant was used for vaccination in BALB/c mice. Vaccination was performed into the left footpad. Three weeks later, the booster was injected in the same manner. To examine the effectiveness of the injected vaccine, pathogenic L. major (MRHO/IR/75/ER) was injected into the right footpad of all mice three weeks following the booster vaccination. In order to assess humoral immunity, the levels of IgG1, and IgG2a antibodies before and 6 weeks after the challenge were studied in the groups. In addition, in order to investigate cellular immunity in the groups, the study measured IFN-γ, IL-5, TNF-α, IL-6 and IL-17 cytokines before, 3 weeks and 8 weeks after the challenge, and also the parasite load in the lymph node with real-time PCR. RESULTS: The lowest level of the parasitic load was observed in the G1 group (mice vaccinated with L. tarentolae-LACK/KMP11/EGFP with CpG) in comparison with other groups (L. tarentolae-LACK/KMP11/EGFP +non-CpG (G2); L. tarentolae-EGFP + CpG (G3, control); L. tarentolae-EGFP + non-CpG (G4, control); and mice injected with PBS (G5, control). Moreover, the evaluation of immune response showed a delayed-type hypersensitivity towards Th1. CONCLUSIONS: According to the results of this study, the live recombinant vaccine of L. tarentolae-LACK/KMP11/EGFP with the CpG adjuvant reduced the parasitic load and footpad induration in infected mice. The long-term effects of this vaccine can be evaluated in volunteers as a clinical trial in future planning.

Activities of artesunate-based combinations and tafenoquine against Babesia bovis in vitro and Babesia microti in vivo.

BACKGROUND: Babesiosis represents a veterinary and medical threat, with a need for novel drugs. Artemisinin-based combination therapies (ACT) have been successfully implemented for malaria, a human disease caused by related parasites, Plasmodium spp. The aim of this study was to investigate whether ACT is active against Babesia in vitro and in vivo. METHODS: Mefloquine, tafenoquine, primaquine, methylene blue and lumefantrine, alone or in combination with artesunate, were tested in vitro against Babesia bovis. Parasite growth was verified using a SYBR green I-based fluorescence assay. Mice infected with Babesia microti were treated with mefloquine or tafenoquine, alone or in combination with artesunate, and parasitemia was verified by microscopy and PCR. RESULTS: All drugs, except lumefantrine, showed in vitro activity against B. bovis, with methylene blue showing the most potent activity (concentration 0.2 µM). Combination with artesunate led to improved activity, with mefloquine showing a striking 20-fold increase in activity. Tafenoquine (10 mg/kg, base), combined or not with artesunate, but not mefloquine, induced rapid clearance of B. microti in vivo by microscopy, but mice remained PCR-positive. Blood from mice treated with tafenoquine alone, but not with tafenoquine-artesunate, was infective for naive mice upon sub-inoculation. CONCLUSIONS: Tafenoquine, and most likely other 8-aminoquinoline compounds, are promising compounds for the development of ACT for babesiosis.

Mimotope-based antigens as potential vaccine candidates in experimental murine cysticercosis.

Human cysticercosis is a public health problem caused by Taenia solium metacestodes; thus, eradication of T. solium transmission by vaccination is an urgent requirement. The Cc48 mimotope from T. solium cysticerci was tested expressed in phage particles (mCc48) and chemically synthesized (sCc48) as a vaccine candidate in experimental murine cysticercosis. For this, BALB/c mice were immunized with mCc48 (G1; n = 40), sCc48 (G2; n = 40) and phosphate-buffered saline (PBS) (G3; n = 40, positive control) and challenged with Taenia crassiceps metacestodes. Another PBS group without parasite challenge was used as a negative control (G4; n = 40). Mice were sacrificed 15, 30, 45 and 60 days post-infection for cysticerci and serum collection. Immunization efficacy was determined by cysticerci counting. Serum samples were tested by ELISA to verify antibody (IgM, IgG, IgA and IgE) and cytokine (IFNγ and IL-4) levels. The sCc48 achieved the highest rates of protection and efficacy (90 and 98%, respectively). The group immunized with mCc48 presented the highest reactivity for IgM, IgG and IgE. All groups presented IL-4, but IFNγ was quite variable among groups. The protection induced by sCc48 synthetic peptide supports further studies of this mimotope as a potential vaccine candidate against cysticercosis.

Impact of clinically acquired miltefosine resistance by Leishmania infantum on mouse and sand fly infection.

OBJECTIVES: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. METHODS: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. RESULTS: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. CONCLUSION: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.

Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection.

BACKGROUND: Different immune mechanisms are capable of killing developmental stages of filarial nematodes and these mechanisms are also likely to vary between the primary and a challenge infection. However, the lack of a detailed analysis of cytokine, chemokine and immunoglobulin levels in human loiasis is still evident. Therefore, detailed analysis of immune responses induced by the different developmental stages of Loa loa in immune-competent BALB/c mice will aid in the characterization of distinct immune responses that are important for the immunity against loiasis. METHODS: Different developmental stages of L. loa were obtained from human peripheral blood (microfilariae, MF), the transmitting vector, Chrysops (larval stage 3, L3) and infected immune-deficient BALB/cRAG2γc-/- mice (L4, L5, adult worms). Groups of wildtype BALB/c mice were then injected with the isolated stages and after 42 days post-infection (pi), systemic cytokine, chemokine and immunoglobulin levels were determined. These were then compared to L. loa-specific responses from in vitro re-stimulated splenocytes from individual mice. All parameters were determined using Luminex technology. RESULTS: In a pilot study, BALB/c mice cleared the different life stages of L. loa within 42 days pi and systemic cytokine, chemokine and immunoglobulin levels were equal between infected and naive mice. Nevertheless, L. loa-specific re-stimulation of splenocytes from mice infected with L5, MF or adult worms led to induction of Th2, Th17 and chemokine secretion patterns. CONCLUSIONS: This study shows that although host immunity remains comparable to naive mice, clearance of L. loa life-cycle development stages can induce immune cell memory leading to cytokine, chemokine and immunoglobulins secretion patterns which might contribute to immunity and protection against reinfection.

Anti-plasmodial effect of plant extracts from Picrolemma huberi and Picramnia latifolia.

BACKGROUND: Malaria is an infectious disease caused by parasites of the genus Plasmodium, of which Plasmodium vivax and Plasmodium falciparum are the major species that cause the disease in humans. As there are relatively few alternatives for malaria treatment, it is necessary to search for new chemotherapeutic options. Colombia possesses a great diversity of plants, which are potential sources of new compounds of medical interest. Thus, in this study the antiplasmodial effect of extracts from two species of plants from the families Simaroubaceae and Picramniaceae (Picramnia latifolia and Picrolemma huberi) was evaluated in vitro and in vivo. These plants were chosen because they contain secondary metabolites with interesting medicinal effects. RESULTS: The ethanolic extracts of both species were highly active with IC50: 1.2 ± 0.19 µg/mL for P. latifolia and IC50: 0.05 ± 0.005 µg/mL for P. huberi. The P. latifolia extract had a stage specific effect on trophozoites and inhibited parasite growth in vivo by 52.1 ± 3.4%, evaluated at 1000 mg/kg in Balb/c mice infected with Plasmodium berghei. On the other hand, evaluated at 150 mg/kg body weight in the same murine model, the ethanolic extract from P. huberi had an antiplasmodial effect in all the asexual intraerythrocytic stages of P. falciparum FCR3 and inhibited the parasitic growth in 93 ± 32.9%. CONCLUSIONS: This is the first report of anti-malarial activity for these two species of plants. Thus, P. latifolia and P. huberi are potential candidates for the development of new drugs for treating malaria.

Comparative Susceptibility of Different Mouse Strains to Liver-Stage Infection With Plasmodium berghei Sporozoites Assessed Using In Vivo Imaging.

BACKGROUND: The liver stages of Plasmodium parasites are important targets for the discovery and development of prophylactic drugs. METHODS: A real-time in vivo imaging system was used to determine the level of luminescence measured from firefly luciferase expression by sporozoites developing in hepatocytes in different strains of mice. RESULTS: The luminescence values (photon counts/sec) measured from the anatomical liver location in the untreated mice infected with 10,000 Plasmodium berghei sporozoites were 8.15 × 105 for C57BL/6 Albino, 2.12 × 105 for C3H/HeNCrL, 0.91 × 105 for C57BL/6 WT, 0.28 × 105 for BALB/c, and 0.16 × 105 for ICR/CD-1 mice. This data suggests that the C57BL/6 Albino strain is most susceptible to luminescent photon, mainly because the less light scattering and absorption from deeper tissues and the skin in the strain of mouse. The photon count observed in black C57BL/6 wild type mice was shown to be 88.83% lower compared to C57BL/6 Albino mice. Although the highest growth rate of sporozoites in hepatocytes was found for C57BL/6 wild type mice in this study, the black skin of this mouse significantly reduced parasite-associated bioluminescence. CONCLUSIONS: The minimal light scattering and absorption and also enhanced susceptibility to liver infection of C57BL/6 Albino mice makes this strain preferable sensitivity for discovery and development of prophylactic antimalarial drugs.

Anti-Leishmanial Activity (In Vitro and In Vivo) of Allicin and Allicin Cream Using Leishmania major (Sub-strain Zymowme LON4) and Balb/c Mice.

BACKGROUND: Leishmania is a unicellular protozoan parasite that produces several human diseases, ranging from localized self-healing cutaneous lesions to deadly visceral infections. OBJECTIVE: The effect of allicin on the growth of Leishmania major (L. major) promastigotes was evaluated under in vitro conditions. Moreover, the efficacy of a topical allicin cream was examined in BALB/c (Bagg albino, laboratory-bred strain of the House Mouse) mice with cutaneous leishmanial lesions compared to the currently used drug, sodiumstibogluconate (pentostam). METHODS: Cytotoxiciy and promastigote proliferation were measured. Different concentrations (50, 100, 150, and 200 µM) of liquid allicin were tested on L. major promastigotes twice: after 24 and 48 hours using an MTT colorimetric assay. In the in vivo condition, the efficacies of allicin cream and liquid allicin at two concentrations (0.15 µM/mouse and 0.30 µM/mouse) were evaluated. Serum factors of the control and treated groups were tested to evaluate the toxic effects of allicin on the liver and kidney. RESULTS: Allicin at a concentration of 50 µM inhibited the growth of Leishmania promastigotes. Topical application of allicin cream reduced lesion sizes in mice. No significant differences in biochemical analysis were observed between the control and treated groups. CONCLUSIONS: Allicin has antileishmanial effects under in vitro and in vivo conditions and may be used in clinical applications.

[Assessment of transmammary transmission of Trichinella patagoniensis in BALB/c mice]. / Evaluación de la transmisión galactógena de Trichinella patagoniensis en ratones BALB/c.

Since 1916 to date, it has been suspected that vertical transmission of parasites from the genus Trichinella could occur in pregnant or lactating women during the parenteral phase of infection. The aim of the present study was to evaluate the transmammary transmission of T. patagoniensis in BALB/c mice. Twenty 7-week-old BALB/c mice were distributed into two groups of 10 individuals each, depending on the time of gestation when they were infected, 15 or 18 days after detection of the vaginal plug. Each group was subdivided into two subgroups of 5 mice each, which were given an oral dose of 100 or 500 infective larvae respectively. Euthanasia and subsequent artificial digestion was performed in the pups and the dams. No T. patagoniensis L1 larvae were found in any of the offsprings analyzed. The observed results suggest that vertical transmission of T. patagoniensis would not be possible in BALB/c mice.

4-Aminoquinoline Derivatives as Potential Antileishmanial Agents.

The leishmanicidal activity of a series of 4-aminoquinoline (AMQ) derivatives was assayed against Leishmania amazonensis. This activity against the intracellular parasite was found stronger than for L. amazonensis promastigotes. Neither compound was cytotoxic against macrophages. The compound AMQ-j, which exhibited a strong activity against promastigotes and amastigotes of L. amazonensis (IC50 values of 5.9 and 2.4 µg/mL, respectively) and similar leishmanicidal activity to reference drugs, was chosen for studies regarding its possible mechanism of action toward parasite death. The results showed that the compound AMQ-j induced depolarization of the mitochondrial membrane potential in promastigotes and in L. amazonensis-infected macrophages, but not in uninfected macrophages. Furthermore, the depolarization of the mitochondrial membrane potential was dose dependent in infected macrophages. We have established that promastigotes and L. amazonensis-infected macrophages treated with AMQ-j were submitted to oxidative stress. This is in line with the increase in the level of reactive oxygen species (ROS). Leishmania amazonensis-infected macrophages treated with AMQ-j did not show a significant increase in the production of nitric oxide. Our results indicate the effective and selective action of AMQ-j against L. amazonensis, and its mechanism of action appears to be mediated by mitochondrial dysfunction associated with ROS production.

Bed bugs (Cimex lectularius) as vectors of Trypanosoma cruzi.

Populations of the common bed bug, Cimex lectularius, have recently undergone explosive growth. Bed bugs share many important traits with triatomine insects, but it remains unclear whether these similarities include the ability to transmit Trypanosoma cruzi, the etiologic agent of Chagas disease. Here, we show efficient and bidirectional transmission of T. cruzi between hosts and bed bugs in a laboratory environment. Most bed bugs that fed on experimentally infected mice acquired the parasite. A majority of previously uninfected mice became infected after a period of cohabitation with exposed bed bugs. T. cruzi was also transmitted to mice after the feces of infected bed bugs were applied directly to broken host skin. Quantitative bed bug defecation measures were similar to those of important triatomine vectors. Our findings suggest that the common bed bug may be a competent vector of T. cruzi and could pose a risk for vector-borne transmission of Chagas disease.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection.

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Monitoramento sanitário de colônia de camundongos BALB/c mantidos em biotério convencional / Health monitoring of the BALB/c mice colony kept in a conventional animal facility

O objetivo deste estudo foi avaliar o estado sanitário da colônia de camundongos BALB/c e as condições ambientais do Biotério do Instituto Lauro de Souza Lima, através de exames parasitológicos e microbiológicos. Utilizando-se meios de cultura enriquecidos e seletivos, procurou-se identificar agentes potencialmente patogênicos para os animais e zoonóticos, a partir de pele, pelos, trato respiratório e entérico dos animais, além da microbiota fúngica ambiental. Pesquisou-se a presença de Mycoplasma sp. e Bordetella sp. em traqueia e fungos em pele e pelos, além de exames parasitológicos e pesquisa de Salmonella spp. e enterobactérias em amostras de conteúdo do ceco dos animais. Para monitoramento do ambiente, coletaram-se amostras de diferentes locais do interior do biotério. Após macro e microcultivo fúngicos, foram identificados Cladosporium sp., Acremonium sp., Aspergillus sp., Curvularia sp. e Trichophyton mentagrophytes. O exame micológico de pele e pelos resultou negativo para 100% das amostras. Não foi observado crescimento de patógenos respiratórios, nem de Salmonella spp. As bactérias isoladas são constituintes da microbiota entérica normal de animais em condições convencionais: Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, Klebsiella spp., Providencia stuart, Pseudomonas putida. O exame parasitológico demonstrou a presença de Strongyloides stercoralis, Syphacia obvelata, Aspiculuris tetraptera e Ancylostoma sp.. O trabalho realizado demonstrou grande valor diagnóstico, tanto para avaliação da saúde dos animais de laboratório, como para uma análise qualitativa da microbiota fúngica ambiental, devendo ser implantado como rotina no biotério.

The aim of this work was to evaluate the health status and environmental conditions of the colony of BALB/c mice maintained in an Animal Facility of the Lauro de Souza Lima Institute. Parasitological and microbiological tests with enriched and selective culture media were used to identify zoonotic and potentially pathogenic agents from skin, hair, respiratory and enteric tracts of the animals and fungal flora of the environment. This study proposed to verify colonization of Mycoplasma sp. and Bordetella sp. in trachea, fungi in skin and hair, as well as perform parasitological examination, Salmonella spp. and enterobacteria isolation from cecum of sampled animals. In addition serum of animals were tested against different viral and bacterial antigens and against Toxoplasma gondii antigen.For environmental monitoring samples were collected from different locations. After macro and microculture, the fungi identified were Cladosporium sp. Acremonium sp., Aspergillus sp., Curvularia sp. and Trichophyton mentagrophytes. Mycological examination of the skin was negative for 100% of the samples. There was no growth of respiratory pathogens or Salmonella spp. The bacteria isolated in the present study consist of the normal enteric microbiota of animals in conventional conditions: Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, Klebsiella spp. Providencia stuart and Pseudomonas putida. The parasitological examination showed the presence of Strongyloides stercoralis, Syphacia obvelata, Aspiculuris tetraptera and Ancylostoma sp. Serology resulted negative for all tested antigens. The techniques used in this work showed to be valuable for diagnostic purpose, to assess the health of laboratory animals, as well as for qualitative analysis of environmental fungal microbiota, thus it should be implemented as a routine in the animal house.

Anopheles stephensi saliva enhances progression of cerebral malaria in a murine model.

Malaria accounts for the greatest morbidity and mortality of any arthropod-borne disease globally. Recently, it was determined that the protective antisporozoite CD8+ T-cell response originates predominantly from cutaneous lymph nodes draining the site of parasite inoculation by an Anopheles mosquito. The female mosquito inoculates sporozoites along with an assortment of salivary proteins into the skin of its mammalian host. Mosquito saliva has demonstrable antihemostatic as well as various immunomodulatory activities, and studies with mosquito-borne viruses support a role for mosquito saliva in enhancement of transmission and exacerbation of disease. Early differences in immune response can be detected, which discriminate between mice that are resistant and susceptible to neurological pathology. This supports the idea that early divergence in the immune response may influence the likelihood of progression to the more severe forms of malaria. To evaluate the effect of mosquito feeding on the pathogenesis and immune response to malaria, we injected washed Plasmodium berghei sporozoites intradermally in the presence or absence of mosquito feeding. We observed that mice exposed to mosquito feeding in tandem with the inoculation of sporozoites had higher parasitemias and an elevated progression to cerebral malaria. This was associated with, in particular, elevated levels of interleukin-4 and interleukin-10, suppression of overall transcription in response to infection, and decreased extravasation of dendritic cells and monocytes. This study enhances to our understanding of the complexity of the interactions between the malaria parasite, its host, and the mosquito vector.

Longevity of Zygocotyle lunata in Balb/c mice and long-term survival of digeneans in vertebrate hosts.

This study used Balb/c mice to examine the longevity of Zygocotyle lunata in a murine host. Of 11 mice, each exposed to 20 Z. lunata cysts, six were infected with a total of 12 worms from 11 to 24 weeks postinfection (PI). Live worms recovered at 24 weeks PI had a mean body area of about 25 mm2. These worms produced viable eggs with well-developed miracidia following embryonation in artificial spring water for 2 weeks at 28°C. The Balb/c mouse is a useful model to study longevity of this paramphistomid trematode for at least 6 months PI. An additional aspect of this article is a review of the pertinent literature published from 1937 to 2007 on ageing and longevity of digeneans.

Interleukin-17-mediated control of parasitemia in experimental Trypanosoma congolense infection in mice.

BALB/c mice are highly susceptible to experimental Trypanosoma congolense infections, whereas C57BL/6 mice are relatively resistant. Infected highly susceptible BALB/c mice die of systemic inflammatory response syndrome. Because interleukin-17 (IL-17) and Th17 cells regulate inflammatory responses, we investigated their role in the pathogenesis of experimental African trypanosomiasis in mice. We show that the production of IL-17 by spleen and liver cells and the serum IL-17 level increased after T. congolense infection in mice. Interestingly, infected highly susceptible BALB/c mice produced more IL-17 and had more Th17 cells than infected relatively resistant C57BL/6 mice. Paradoxically, neutralization of IL-17 with anti-IL-17 monoclonal antibody in vivo induced higher parasitemia in both the susceptible and the relatively resistant mice. Interestingly, anti-IL-17 antibody-treated mice had higher serum levels of alanine aminotransferase and aspartate aminotransferase, and the production of IL-10 and nitric oxide by liver cells was markedly decreased. Moreover, recombinant IL-17-treated mice exhibited significantly faster parasite control and lower peak parasitemia compared to control mice. Collectively, these results suggest that the IL-17/Th17 axis plays a protective role in murine experimental African trypanosomiasis.

Suppressing glucose transporter gene expression in schistosomes impairs parasite feeding and decreases survival in the mammalian host.

Adult schistosomes live in the host's bloodstream where they import nutrients such as glucose across their body surface (the tegument). The parasite tegument is an unusual structure since it is enclosed not by the typical one but by two closely apposed lipid bilayers. Within the tegument two glucose importing proteins have been identified; these are schistosome glucose transporter (SGTP) 1 and 4. SGTP4 is present in the host interactive, apical tegumental membranes, while SGTP1 is found in the tegumental basal membrane (as well as in internal tissues). The SGTPs act by facilitated diffusion. To examine the importance of these proteins for the parasites, RNAi was employed to knock down expression of both SGTP genes in the schistosomula and adult worm life stages. Both qRT-PCR and western blotting analysis confirmed successful gene suppression. It was found that SGTP1 or SGTP4-suppressed parasites exhibit an impaired ability to import glucose compared to control worms. In addition, parasites with both SGTP1 and SGTP4 simultaneously suppressed showed a further reduction in capacity to import glucose compared to parasites with a single suppressed SGTP gene. Despite this debility, all suppressed parasites exhibited no phenotypic distinction compared to controls when cultured in rich medium. Following prolonged incubation in glucose-depleted medium however, significantly fewer SGTP-suppressed parasites survived. Finally, SGTP-suppressed parasites showed decreased viability in vivo following infection of experimental animals. These findings provide direct evidence for the importance of SGTP1 and SGTP4 for schistosomes in importing exogenous glucose and show that these proteins are important for normal parasite development in the mammalian host.

Comparison of the pathogensis of two isolates of Besnoitia caprae in inbred BALB/c mice.

This study was performed to evaluate the infectivity of bradyzoites of two Besnoitia caprae isolates, BC-1 and BC-2, to inbred BALB/c mice. Each group of inbred BALB/c mice was inoculated intraperitoneally with 1 x 10(3), 1 x 10(4), 1 x 10(5), 5 x 10(5) and 1 x 10(6) of one of the two isolates of B. caprae bradyzoites. The mice were monitored daily for a period of 40 days for survival. After death of each mice, several passages from its peritoneal washing and tissues were analyzed using ribosomal DNA-specific PCR assay. Marked differences in pathogenicity between the isolates were seen. All the inbred BALB/c mice infected with BC-2 survived but all the mice that were administered with 1 x 0(5), 5 x 10(5) and 1 x 10(6) BC-1 bradyzoites were died within 4-9 days post-infection (DPI). Histopathological examination of the tissues of the dead mice revealed hyperemia and necrosis with presence of mononuclear and polymorphonuclear cell infiltration in myocardium, spleen and intestines together with interstitial pneumonia and peritonitis. All inbred BALB/c mice in the 1 x 10(3) and 1 x 10(4) groups of BC-1 inoculated mice survived and they were euthanized after 40 DPI. Chronic inflammation with infiltration of mononuclear cells was evident in myocardium, spleen, alveolar septa of the lungs of most of the examined tissues with hemorrhagic enteritis in the mice infected with 1 x 10(6) bradyzoites. The mice infected with different doses of BC-2 were euthanized after 40 DPI and no lesion was seen in histopathological sections of their organs. All peritoneal washings and examined tissues were PCR positive in BC-1 group. This experiment is the first report to show inbred BALB/c mice as a relevant model for B. caprae and demonstrates that this strain of inbred BALB/c mice is a suitable animal model for biological studies and examination of pathogenesis for this species of Besnoitia. The present findings also provide evidence for significant differences between the two isolates of B. caprae.

Differential in vitro effects of insulin on Taenia crassiceps and Taenia solium cysticerci.

Hormones play a significant role in murine cysticercosis (Taenia crassiceps), and increase the frequency of porcine cysticercosis caused by Taenia solium. In the present study, we report the in vitro effect of insulin on the larval stages of T. crassiceps (ORF strain) and T. solium. In vitro exposure of T. crassiceps cysticerci to insulin was found to stimulate this parasite's reproduction twofold with respect to control values, while the same treatment had no effect on T. solium cysticerci. Moreover, normal female mice (BALB/cAnN) infected with T. crassiceps cysticerci previously exposed to insulin presented larger parasite loads than mice inoculated with vehicle-treated cysticerci. To determine the possible molecular mechanisms by which insulin affects T. crassiceps, the insulin receptor was amplified by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Interestingly, both T. crassiceps and T. solium expressed the insulin receptor, although insulin had effects only on T. crassiceps. These results demonstrate that insulin has a dichotomistic effect; it acts directly only on T. crassiceps cysticerci reproduction, possibly through its binding to a specific insulin receptor synthesized by the parasite. Thus, insulin may be recognized by T. crassiceps cysticercus cells as a mitogenic factor, and contribute to parasite proliferation inside the host, as well as to the female mouse susceptibility to T.crassiceps. This phenomenon has not been reported for cysticercosis caused by T. solium, which could, in part, be related to the poor effect of insulin upon the human parasite.