Morphology and Molecular Identification of Echinostoma revolutum and Echinostoma macrorchis in Freshwater Snails and Experimental Hamsters in Upper Northern Thailand.

Echinostome metacercariae were investigated in freshwater snails from 26 districts in 7 provinces of upper northern Thailand. The species identification was carried out based on the morphologies of the metacercariae and adult flukes harvested from experimental hamsters, and on nucleotide sequences of internal transcribed spacer 2 (ITS2) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) genes. Twenty-four out of 26 districts were found to be infected with echinostome metacercariae in freshwater snails with the prevalence of 40.4%. The metacercariae were found in all 6 species of snails, including Filopaludina martensi martensi (21.9%), Filopaludina doliaris (50.8%), F. sumatrensis polygramma (61.3%), Bithynia siamensis siamensis (14.5%), Bithynia pulchella (38.0%), and Anenthome helena (4.9%). The echinostome metacercariae found in these snails were identified as Echinostoma revolutum (37-collar-spined) and Echinostoma macrorchis (45-collar-spined) morphologically and molecularly. The 2-week-old adult flukes of E. revolutum revealed unique features of the cirrus sac extending to middle of the ventral sucker and smooth testes. E. macrorchis adults revealed the cirrus sac close to the right lateral margin of the ventral sucker and 2 large and elliptical testes with slight indentations and pointed posterior end of the posterior testis. The ITS2 and nad1 sequences confirmed the species identification of E. revolutum, and the sequences of E. macrorchis have been deposited for the first time in Gen-Bank. The presence of the life cycle of E. macrorchis is a new record in Thailand and the snail F. doliaris as their second intermediate host seems to be new among the literature.

The hidden faces of a biological invasion: parasite dynamics of invaders and natives.

One of the primary drivers of emerging infectious diseases (EIDs) is human intervention via host or parasite translocations. A unique opportunity to study host and parasite dispersal during a bio-invasion currently exists in Ireland due to the introduction of the bank vole (Myodes glareolus) in the 1920s. The continuing range expansion of M. glareolus within Ireland presents a natural large-scale perturbation experiment. This study used the Irish M. glareolus model to conduct a spatiotemporal study analysing the parasite dynamics of native and invasive species throughout their range. Myodes glareolus and native Apodemus sylvaticus were trapped in woodlands across Ireland and surveyed for their helminth parasites. Myodes glareolus in Ireland were found to have lower parasite diversity in comparison to records of M. glareolus from across Europe and A. sylvaticus in Ireland. Increased density of M. glareolus resulted in a dilution effect, with significantly lower levels of parasitism overall in native hosts, where M. glareolus has been established longest. However, three helminth parasite species of A. sylvaticus increased in abundance in the presence of M. glareolus. Furthermore, M. glareolus at the expansion front were less parasitised (lower abundance and prevalence of certain parasites and lower parasite diversity) than M. glareolus from the core population. This "enemy release" is believed to be mediating the continued successful spread of the invader across Ireland. Our results identify two important variables, seasonality and the stage of the invasion, which should not be overlooked when investigating or managing the changing distribution of hosts and their parasites. Studies of bio-invasions and parasite transmission have primarily focused on the invasive host species or the native host species in cases where virulent pathogen spillover is observed. Our results demonstrate how the concurrent study of invasive and native hosts, and the careful identification of their parasite communities, allows the dynamic processes influencing the parasite component and intracommunity to be identified.

Effectiveness of Repeated Administration of Praziquantel with Disodium Glycyrrhizinate and Two Enantiomers of Praziquantel on Opisthorchis felineus (Rivolta, 1884).

BACKGROUND: Nowadays, it is still important to develop effective anti-opisthorchiasis agents. In this work, we tested a complex of praziquantel (PZQ) with a plant origin compound-disodium glycyrrhizinate-in the ratio 1:10 PZQ:Na2GA, containing 11-fold less of the active ingredient. Our aim was to study various ways to treat trematode Opisthorchis felineus with this complex in vitro. Additionally, an in vitro comparison of the anthelmintic action was made among racemic-PZQ, (R)-PZQ, and (S)-PZQ on juvenile and adult maritae of O. felineus. METHODS: Worms extracted from the hamsters were subjected to various regimens of administration of the complex: once a day for 3 days or three times within 1 day. Moreover, mature maritae and juvenile worms of O. felineus were subjected to the comparison the anthelmintic effectiveness of racemic-PZQ, (R)-PZQ, and (S)-PZQ. RESULTS: The O. felineus maritae that received PZQ:Na2GA (1:10) thrice within 1 day were most strongly affected by the drug. Their motility substantially decreased already on the second day after the last dose, and the percentage of live worms by the end of the experimental period was the lowest. These results indicate a cumulative anthelmintic effect of this substance under the regimen "three times within 1 day." For the first time, we report that among the three substances (racemic-PZQ and two enantiomers), (R)-PZQ has the highest anthelmintic activity, toward both juvenile and sexually mature maritae of O. felineus. CONCLUSION: These findings suggest that the development of a supramolecular complex of (R)-PZQ with disodium glycyrrhizinate and administration of this complex three times within 1 day are promising approaches.

Oxyurid nematodes of pet rodents in Slovakia - a neglected zoonotic threat / Nematódeos oxiurídeos de roedores de estimação na Eslováquia - uma ameaça zoonótica negligenciada

Abstract The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.

Resumo O papel dos roedores como reservatórios de helmintos de importância para a saúde pública não é bem conhecido. O potencial zoonótico de Syphacia spp. foi confirmado; portanto, o estudo teve como objetivo estimar a ocorrência de nematóides oxiurídeos em pequenos roedores de pet shops e clubes de reprodução na Eslováquia. Amostras fecais de 586 roedores mantidos em 133 gaiolas foram coletadas entre 2016 e 2018 e examinadas pelo método de flotação de Faust. Foram detectadas quatro espécies de nematódeos oxiurídeos, Syphacia muris, S. obvelata, Aspiculuris tetraptera e Paraspidodera uncinata, A. tetraptera foi encontrado nas amostras fecais de todas as espécies de roedores incluídas nesta pesquisa. O número de gaiolas positivas variou de 5,4% em hamsters a 70,0% em camundongos. A prevalência de Syphacia muris foi maior nos gerbilos da Mongólia, onde até 75,0% das gaiolas foram positivas; S. obvelata foi encontrada em 26,7% das gaiolas com camundongos, 25,0% das gaiolas com gerbilos da Mongólia e 3,2% das gaiolas com ratos. A alta prevalência de Syphacia spp. em todas as espécies animais aponta o risco de infecção para os seres humanos. Animais oferecidos para venda estão frequentemente em contato próximo com seres humanos; portanto, eles devem ser regularmente testados quanto a parasitas e, então, efetivamente desparasitados.

Oxyurid nematodes of pet rodents in Slovakia - a neglected zoonotic threat.

The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.

New insights from Opisthorchis felineus genome: update on genomics of the epidemiologically important liver flukes.

BACKGROUND: The three epidemiologically important Opisthorchiidae liver flukes Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, are believed to harbour similar potencies to provoke hepatobiliary diseases in their definitive hosts, although their populations have substantially different ecogeographical aspects including habitat, preferred hosts, population structure. Lack of O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease. RESULTS: Here we present the first draft genome assembly of O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis. We observed both noticeably high heterozygosity of the sequenced individual and substantial genetic diversity in a pooled sample. This indicates that potency of O. felineus population for rapid adaptive response to control and preventive measures of opisthorchiasis is higher than in O. viverrini and C. sinensis. We also have found that all three species are characterized by more intensive involvement of trans-splicing in RNA processing compared to other trematodes. CONCLUSION: All revealed peculiarities of structural organization of genomes are of extreme importance for a proper description of genes and their products in these parasitic species. This should be taken into account both in academic and applied research of epidemiologically important liver flukes. Further comparative genomics studies of liver flukes and non-carcinogenic flatworms allow for generation of well-grounded hypotheses on the mechanisms underlying development of cholangiocarcinoma associated with opisthorchiasis and clonorchiasis as well as species-specific mechanisms of these diseases.

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.

Prevalence and zoonotic risk of tropical rat mite (Ornithonyssus bacoti) in exotic companion mammals in southern Italy.

BACKGROUND: Exotic companion mammals are popular pets worldwide. They are a potential source of zoonotic infections transmissible to their owners. HYPOTHESIS/OBJECTIVES: To determine the prevalence and zoonotic risks of tropical rat mite (Ornithonyssus bacoti) in exotic companion mammals in Italy. ANIMALS: The records of 782 exotic pet mammals seen in multiple veterinary clinics (n = 20), pet shops (n = 10) and private breeders (n = 2) around Naples (Italy) were searched. METHODS AND RESULTS: The isolation of O. bacoti was the only inclusion criterion. Relative (in the subgroups) and absolute prevalence (in the entire population sampled) of clinical signs in pets and owners were calculated. The prevalence of clinical signs in pets and their owners was also calculated based on their housing (pet shops versus private housing) using Fisher's exact test. A P-value < 0.05 was considered significant. Seventy seven records (9.8%) of animals infested were identified. Of those, 33.8% (26 of 77) were hamsters, 25.9% (20 of 77) gerbils, 11.7% (nine of 77) guinea pigs, 7.8% (six of 77) rabbits, 7.8% (six of 77) degus, 5.2% (four of 77) kangaroo mice, 2.6% (two of 77) hedgehogs, 2.6% (two of 77) squirrels and 2.6% (two of 77) were sugar gliders. The frequency of owners affected by the rat mite dermatitis was very high in gerbils (20 of 20), hamsters (21 of 26) and guinea pigs (seven of nine). CONCLUSIONS AND CLINICAL IMPORTANCE: The results of the present survey indicate that exotic pet mammals may serve as an active reservoir for O. bacoti infestation. The results of this study also suggest a lack of species specificity for O. bacoti when favourable conditions are present (overcrowding).

Increased Transmissibility of Leishmania donovani From the Mammalian Host to Vector Sand Flies After Multiple Exposures to Sand Fly Bites.

Background: Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. The role of the blood or skin as a source of infection to sand flies remains unclear, and the possible effect of multiple exposures to fly bites on transmissibility has not been addressed. Methods: L. donovani-infected hamsters underwent xenodiagnoses with Lutzomyia longipalpis on the same or different sites on the abdomen on 2 consecutive days or by artificial feeding on the skin or blood. Results: The transmission of L. donovani from sick hamsters to flies was surprisingly low (mean, 24% of fed flies). New flies fed on the same site acquired significantly more infections (mean, 61%; P < .0001). By artificial feeding, flies could acquire infection from blood and skin. However, only artificial feeding on blood produced infections that correlated with the natural feeding (R = 0.792; P < .0001). Infections acquired from blood increased dramatically for blood obtained after exposure to bites, as did the parasitemia level and the number of monocytes in the circulation. Conclusions: The bites of uninfected sand flies favor the transmissibility of L. donovani by infected hosts, owing to a systemic effect that exposure to bites has on the parasitemia. Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. Using the hamster model of visceral disease, we demonstrate that prior exposure to bites of uninfected sand flies potentiates their ability to transmit infection to the vector.

Screening for Babesia microti in the U.S. Blood Supply.

BACKGROUND: Babesia microti, a tickborne intraerythrocytic parasite that can be transmitted by means of blood transfusion, is responsible for the majority of cases of transfusion-transmitted babesiosis in the United States. However, no licensed test exists for screening for B. microti in donated blood. We assessed data from a large-scale, investigational product-release screening and donor follow-up program. METHODS: From June 2012 through September 2014, we performed arrayed fluorescence immunoassays (AFIAs) for B. microti antibodies and real-time polymerase-chain-reaction (PCR) assays for B. microti DNA on blood-donation samples obtained in Connecticut, Massachusetts, Minnesota, and Wisconsin. We determined parasite loads with the use of quantitative PCR testing and assessed infectivity by means of the inoculation of hamsters and the subsequent examination for parasitemia. Donors with test-reactive samples were followed. Using data on cases of transfusion-transmitted babesiosis, we compared the proportions of screened versus unscreened donations that were infectious. RESULTS: Of 89,153 blood-donation samples tested, 335 (0.38%) were confirmed to be positive, of which 67 (20%) were PCR-positive; 9 samples were antibody-negative (i.e., 1 antibody-negative sample per 9906 screened samples), representing 13% of all PCR-positive samples. PCR-positive samples were identified all through the year; antibody-negative infections occurred from June through September. Approximately one third of the red-cell samples from PCR-positive or high-titer AFIA-positive donations infected hamsters. Follow-up showed DNA clearance in 86% of the donors but antibody seroreversion in 8% after 1 year. In Connecticut and Massachusetts, no reported cases of transfusion-transmitted babesiosis were associated with screened donations (i.e., 0 cases per 75,331 screened donations), as compared with 14 cases per 253,031 unscreened donations (i.e., 1 case per 18,074 unscreened donations) (odds ratio, 8.6; 95% confidence interval, 0.51 to 144; P=0.05). Overall, 29 cases of transfusion-transmitted babesiosis were linked to blood from infected donors, including blood obtained from 10 donors whose samples tested positive on the PCR assay 2 to 7 months after the implicated donation. CONCLUSIONS: Blood-donation screening for antibodies to and DNA from B. microti was associated with a decrease in the risk of transfusion-transmitted babesiosis. (Funded by the American Red Cross and Imugen; ClinicalTrials.gov number, NCT01528449 .).

Evaluation of 17-AAG as a chemotherapeutic potential for the treatment of visceral leishmaniasis in experimentally infected hamsters / Evaluation of 17-AAG as a chemotherapeutic potential for the treatment of visceral leishmaniasis in experimentally infected hamsters

A leishmaniose é causada por protozoários do gênero Leishmania. Considerada endêmica em diversos países, incluindo o Brasil, o tratamento dessa doença consiste na utilização de agentes quimioterápicos. No entanto, esses fármacos apresentam diversos efeitos colaterais, alto custo e aumento progressivo de falha terapêutica com baixas taxas de cura. Tendo em vista a necessidade para desenvolvimento de novos compostos anti-Leishmania, ensaios realizados anteriormente por nosso grupo evidenciaram o potencial do 17 - allylamino 17 - demethoxygeldanamycin (17-AAG) no tratamento in vivo da leishmaniose cutânea (LC). Esse composto inibe a atividade ATPásica da proteína de choque térmico 90 (HSP90), que no parasito é conhecida por exercer funções essenciais. Como consequência, sua inibição pode levar à morte do parasito, resultando no controle da doença. Assim, nosso estudo avaliou o potencial do 17-AAG no tratamento da leishmaniose visceral (LV) utilizando hamster como modelo experimental. Este modelo foi utilizado, pois apresenta características clínico-patológicas semelhantes às observadas em humanos com calazar. Inicialmente foi realizado um ensaio de toxicidade com o 17-AAG utilizando doses intraperitoneais de 20 ou 40 mg/kg diariamente ou em dias alternados, totalizando quinze aplicações. O esquema terapêutico causou toxicidade no fígado dos animais tratados com ambas às doses e no rim dos animais tratados com 40mg/kg. Todos os animais que receberam 40mg/kg e 80% daqueles que receberam 20mg/kg vieram a óbito no final dos dois esquemas terapêutico. Com o intuito de reduzir a toxicidade e avaliar o potencial leishmanicida do 17-AAG, experimentos subsequentes foram realizados utilizando doses menores de 5 ou 10 mg/kg de 17-AAG em intervalos maiores de três dias, totalizando sete aplicações. Os animais do estudo apresentavam alterações bioquímicas e hematológicas, antes mesmo de iniciar o tratamento, permanecendo com essas alterações durante todos os períodos avaliados, que foram igualmente observadas nos animais do grupo controle não tratados com 17-AAG. Com o intuito de avançar na avaliação de toxicidade e eficácia do tratamento, outro ensaio foi realizado utilizando doses de 10 ou 20 mg/kg de 17-AAG em dias alternados, totalizando sete aplicações. A análise histopatológica mostrou que os hamsters tratados não apresentaram alterações em fígado e rim adicionais às observadas no grupo controle. Esses achados mostram que o tratamento com 17-AAG foi tóxico somente para as células hepáticas dos animais, quando aplicado em doses mais elevadas e em intervalos de tempos curtos. Em doses mais baixas e intervalos maiores, as análises histopatológicas do fígado e rim dos animais revelaram alterações similares às causadas pelo diluente. Quando os hamsters foram tratados com as doses toleradas de 10 ou 20 mg/kg de 17-AAG em dias alternados, o 17-AAG não foi capaz de reduzir a carga parasitária no baço, fígado ou linfonodo desses animais. Em conjunto, esses dados mostram que os hamsters não representam um modelo adequado para teste com 17-AAG, pois os mesmos não toleraram doses elevadas deste fármaco que seriam necessárias para o controle da infecção, indicando a necessidade da utilização de outro modelo experimental nos estudos de tratamento da LV

Survey of Hymenolepis spp. in pet rodents in Italy.

We carried out the first survey of Hymenolepis spp. infection in pet rodents in Italy. Fresh fecal samples were collected from 172 pet rodents as follows: guinea pigs (Cavia porcellus; n = 60), squirrels (Callosciurus finlaysonii, Callosciurus prevosti, Tamias striatus, Tamias sibiricus, Sciurus calorinensis; n = 52), hamsters (Phodopus campbelli, Mesocricetus auratus; n = 30), chinchillas (Chinchilla lanigera; n = 13), rats (Rattus norvegicus; n = 10), and mice (Mus minutoides; n = 7). These animals were housed either in pet shops or in private houses. All fecal samples were processed using the FLOTAC pellet technique to assess the number of eggs per gram (EPG) of feces. Eggs of Hymenolepis nana were found in 24 out of 172 (13.9 %; 95 % confidence interval = 9.3-20.2 %) pet rodents. Of those rodents, 41.6 % (10/24) were rats (mean EPG = 55.7; range = 2-200), 29.2 % (7/24) mice (mean EPG = 64.5; range = 32-120), 25.0 % (6/24) were chinchillas (mean EPG = 25.5; range = 10-50), and 4.2 % (1/24) hamsters (P. campbelli) (EPG = 86.0). In addition, Hymenolepis diminuta eggs were found in 2 out of 172 (1.2 %; 95 % confidence interval = 0.2-4.6 %) rodents examined, both of which (100 %; 2/2) were pet squirrels (C. prevosti) (mean EPG = 10; range = 4-16). To the authors' knowledge, this is the first report of a natural infection of H. diminuta in pet squirrels.

Protection and Delivery of Anthelmintic Protein Cry5B to Nematodes Using Mesoporous Silicon Particles.

The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed in vitro and in vivo. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 µm size are noticeably ingested by both species within 2 h of introduction in vitro, whereas 5 ± 2 µm particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 µm display the highest level of in vitro toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. In vivo experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal.

Pharmacological assessment defines Leishmania donovani casein kinase 1 as a drug target and reveals important functions in parasite viability and intracellular infection.

Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.

Experimental final hosts of Metagonimus hakubaensis (Trematoda: Heterophyidae) and their suitability to the fluke.

Seven laboratory mammal and bird species were orally inoculated with 200-1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.

Vaccination with leishmania hemoglobin receptor-encoding DNA protects against visceral leishmaniasis.

Leishmaniasis is a severe infectious disease. Drugs used for leishmaniasis are very toxic, and no vaccine is available. We found that the hemoglobin receptor (HbR) of Leishmania was conserved across various strains of Leishmania, and anti-HbR antibody could be detected in kala-azar patients' sera. Our results showed that immunization with HbR-DNA induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and hamsters. Moreover, HbR-DNA immunization stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) with concomitant down-regulation of disease-promoting cytokines like IL-10 and IL-4. HbR-DNA vaccination also induced a protective response by generating multifunctional CD4(+) and CD8(+) T cells. All HbR-DNA-vaccinated hamsters showed sterile protection and survived during an experimental period of 8 months. These findings demonstrate the potential of HbR as a vaccine candidate against visceral leishmaniasis.

Oral treatment of rodents with fipronil for feed-through and systemic control of sand flies (Diptera: Psychodidae).

The sand fly Phlebotomus papatasi Scopoli is the vector of Leishmania major (Yakimoff & Schokhor), which is maintained in populations of burrowing rodents. The purpose of this study was to conduct a laboratory study to determine the efficacy of oral treatment of rodents with fipronil for control of sand flies that feed on rodent feces as larvae or on rodent blood as adults. We determined through larval bioassays that fipronil was eliminated in feces of orally-treated hamsters at a level that was significantly toxic to sand fly larvae for 21 d after the hamsters had been withdrawn from a fipronil-treated diet. Through bloodfeeding bioassays, we also found that fipronil was present in the peripheral blood of hamsters at a concentration that was significantly toxic to bloodfeeding adult female sand flies for 49 d after the hamsters had been withdrawn from their treated diet. The results of this study suggest that fipronil acts as well as or better than feed-through or systemic insecticides that previously have been measured against sand flies, and is particularly promising because this single compound acts against both larvae and bloodfeeding adults. An area-wide approach using rodent baits containing a fipronil could suppress vector populations that originate in the vicinity of rodent reservoirs, and could be used to eliminate the most epidemiologically important part of the vector population: female sand flies that take bloodmeals on rodent reservoirs.

Avaliação das histonas de L. infantum como candidatos à vacinas na infecção por l.infantum em hamsters / Evaluation of histones from L. infantum as candidate vaccines in infection in hamsters l.infantum

A leishmaniose visceral é uma doença infecciosa grave, causada por protozoários intracelulares obrigatórios do gênero Leishmania. Vários antígenos de Leishmania têm sido avaliados como candidatos vacinais, destacando-se as proteínas de histonas (HIS), antígenos altamente conservados. A exposição de HIS pela Leishmania induz uma resposta imune potente no hospedeiro vertebrado. Desse modo, neste estudo, avaliamos, em hamsters, a capacidade imunoprotetora dos antígenos de histonas contra a infecção por Leishmania infantum. Os animais foram vacinados com estratégia homóloga, utilizando-se plasmídeos de DNA que codificam para HIS (pcDNA3LiH2A-H3, pcDNA3LiH2B-H4) ou heteróloga (DNA/proteínas HIS) mais 1nM de CpG. Quinze dias após a última imunização, os animais foram desafiados pela via intradérmica com 105 Leishmania infantum metacíclicas mais 0,5 par de glândula salivar de Lutzomya longipalpis. Após a última imunização e durante a infecção, realizaram-se dosagens de citocinas por PCR em tempo real (linfonodo e baço), sorologia por ELISA (soro), carga parasitária por diluição limitante e análise histopatológica de tecidos (linfonodo, baço e fígado). Detectou-se produção de anticorpos IgG anti HIS nos grupos imunizados com a estratégia homóloga e heteróloga, quando comparados aos hamsters não imunizados. As imunizações homóloga e heteróloga diferiram na razão IFN-γ/IL-10 no linfonodo em relação ao grupo controle. Não houve diferença significativa na expressão dessas citocinas no baço após a imunização, entretanto, cinco meses após o desafio o grupo homólogo apresentou um aumento na produção de IL-10 nesse órgão. Na análise histopatológica do baço, verificou-se formação de mais folículos com centro germinativo, evidentes nos animais imunizados independentemente do grupo analisado. Observou-se, também, leucocitose intrasinusoidal e periportal no fígado, e folículos reativos no linfonodo. Nenhuma das estratégias de imunizações com antígenos de histonas acarretou em diminuição da carga parasitária no linfonodo, baço e fígado. As estratégias de imunização homóloga e heteróloga, com antígenos de histonas nucleossomais, não foram capazes de proteger contra infecção por L. infantum no modelo do hamster.

Parasite burden in hamsters infected with two different strains of leishmania (Leishmania) infantum: "Leishman Donovan units" versus real-time PCR.

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum's route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.

MCR synthesis of praziquantel derivatives.

Schistosomiasis, a high volume neglected tropical disease affecting more than 200 million people worldwide, can only be effectively treated by the tetrahydroisoquinoline drug praziquantel (PZQ). Herein, we describe an efficient approach to access PZQ derivatives by the Ugi 4-component reaction followed by the Pictet-Spengler reaction in a two-step, one-pot procedure. 30 novel PZQ derivatives are described based on the Ugi 4-component reaction and an X-ray structure of a novel derivative revealing different conformation compared with PZQ is discussed. Several analogues comparable in activity to the drug PZQ have been identified based on an in vitro Schistosoma mansoni worm viability assay.