Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil

Abstract Background Extra-Amazonian autochthonous Plasmodium vivax infections have been reported in mountainous regions surrounded by the Atlantic Forest in Espírito Santo state, Brazil. Methods Sixty-five patients and 1,777 residents were surveyed between April 2001 and March 2004. Laboratory methods included thin and thick smears, multiplex-PCR, immunofluorescent assay (IFA) against P. vivax and Plasmodium malariae crude blood-stage antigens and enzyme-linked immunosorbent assay (ELISA) for antibodies against the P. vivax-complex (P. vivax and variants) and P. malariae/Plasmodium brasilianum circumsporozoite-protein (CSP) antigens. Results Average patient age was 35.1 years. Most (78.5%) were males; 64.6% lived in rural areas; 35.4% were farmers; and 12.3% students. There was no relevant history of travel. Ninety-five per cent of the patients were experiencing their first episode of malaria. Laboratory data from 51 patients were consistent with P. vivax infection, which was determined by thin smear. Of these samples, 48 were assayed by multiplex-PCR. Forty-five were positive for P. vivax, confirming the parasitological results, while P. malariae was detected in one sample and two gave negative results. Fifty percent of the 50 patients tested had IgG antibodies against the P. vivax-complex or P. malariae CSP as determined by ELISA. The percentages of residents with IgM and IgG antibodies detected by IFA for P. malariae, P. vivax and Plasmodium falciparum who did not complain of malaria symptoms at the time blood was collected were 30.1% and 56.5%, 6.2% and 37.7%, and 13.5% and 13%, respectively. The same sera that reacted to P. vivax also reacted to P. malariae. The following numbers of samples were positive in multiplex-PCR: 23 for P. vivax; 15 for P. malariae; 9 for P. falciparum and only one for P. falciparum and P. malariae. All thin and thick smears were negative. ELISA against CSP antigens was positive in 25.4%, 6.3%, 10.7% and 15.1% of the samples tested for "classical" P. vivax (VK210), VK247, P. vivax-like and P. malariae, respectively. Anopheline captures in the transmission area revealed only zoophilic and exophilic species. Conclusion The low incidence of malaria cases, the finding of asymptomatic inhabitants and the geographic separation of patients allied to serological and molecular results raise the possibility of the existence of a simian reservoir in these areas.

Assessing malaria transmission in a low endemicity area of north-western Peru

Background Where malaria endemicity is low, control programmes need increasingly sensitive tools for monitoring malaria transmission intensity (MTI) and to better define health priorities. A cross-sectional survey was conducted in a low endemicity area of the Peruvian north-western coast to assess the MTI using both molecular and serological tools. Methods Epidemiological, parasitological and serological data were collected from 2,667 individuals in three settlements of Bellavista district, in May 2010. Parasite infection was detected using microscopy and polymerase chain reaction (PCR). Antibodies to Plasmodium vivax merozoite surface protein-119 (PvMSP119) and to Plasmodium falciparum glutamate-rich protein (PfGLURP) were detected by ELISA. Risk factors for exposure to malaria (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific antibody prevalence of both P. falciparum and P. vivax were analysed using a previously published catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR). Results The overall parasite prevalence by microscopy and PCR were extremely low: 0.3 and 0.9%, respectively for P. vivax, and 0 and 0.04%, respectively for P. falciparum, while seroprevalence was much higher, 13.6% for P. vivax and 9.8% for P. falciparum. Settlement, age and occupation as moto-taxi driver during previous year were significantly associated with P. falciparum exposure, while age and distance to the water drain were associated with P. vivax exposure. Likelihood ratio tests supported age seroprevalence curves with two SCR for both P. vivax and P. falciparum indicating significant changes in the MTI over time. The SCR for PfGLURP was 19-fold lower after 2002 as compared to before (λ1 = 0.022 versus λ2 = 0.431), and the SCR for PvMSP119 was four-fold higher after 2006 as compared to before (λ1 = 0.024 versus λ2 = 0.006). Conclusion Combining molecular and serological tools considerably enhanced the capacity of detecting current and past exposure to malaria infections and related risks factors in this very low endemicity area. This allowed for an improved characterization of the current human reservoir of infections, largely hidden and heterogeneous, as well as providing insights into recent changes in species specific MTIs. This approach will be of key importance for evaluating and monitoring future malaria elimination strategies.

Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and Elisa tests for the diagnosis of Chagas disease

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Intravital microscopy technique to study parasite dynamics in the labyrinth layer of the mouse placenta

Intravital imaging techniques are the best approach to investigate in situ cellular behavior under physiological conditions. Many techniques have emerged during these last few years for this purpose. We recently described an intravital imaging technique that allows for the observation of placenta physiological responses at the labyrinth layer of this tissue. This technique will be very useful to study many placental opportunistic infections and in this article we reinforce its usefulness by analyzing placental physiological entrapment of beads and parasites. In particular, our results show that small beads (1.0 μm) or Plasmodium chabaudi-GFP-infected-Red Blood Cells (Pc-GFP-iRBCs) cannot get trapped inside small or large blood vessels of popliteal lymph nodes (PLNs). Inside the placenta, clusters of beads could only be found inside the maternal blood vessels. However, Pc-GFP-iRBCs were found inside and outside the maternal blood vessels. We observed that trophoblasts can ingest infected-Red Blood Cells (iRBCs) in vitro and immunofluorescence of placenta revealed Pc-GFP-iRBCs inside and outside the maternal blood vessels. Taken together, we conclude that fast deposition of particles inside blood vessels seems to be an intrinsic characteristic of placenta blood flow, but iRBCs could be internalized by trophoblast cells. Thus these results represent one of the many possible uses of our intravital imaging technique to address important questions inside the parasitological field.

Stato sanitario di Spigole (Dicentrarchus Labrax) ed Orate (Sparus Aurata) allevate in Grecia e in Italia in relazione alla presenza di agenti di Zoonosi ed Ectoparassiti patogeni

Le attività di ricerca della presente tesi di dottorato si sono focalizzata principalmente sulla parassitofauna dei pesci marini allevati in Grecia ed in Italia con particolare attenzione allo studio degli ectoparassiti di maggior rilievo sanitario in maricoltura ed alla ricerca di endoparassiti di potenziale interesse zoonosico, in particolare larve di nematodi Anisakidae del genere Anisakis. Nel corso del triennio sono stati sottoposti ad esami parassitologici 916 spigole (Dicentrarchus labrax) e 462 orate (Sparus aurata) prelevate presso diverse tipologie di allevamenti greci ed italiani. Per quanto concerne le spigole, la presenza di ectoparassiti è stata riscontrata nel 29,2% e nel 61,9% dei soggetti provenienti rispettivamente da impianti siti in Grecia ed in Italia, mentre le orate hanno presentato percentuali di positività rispettivamente del 87,5% e del 26,7%. Gli ectoparassiti dominanti sono risultati essere il monogeneo Diplectanum aequans nelle spigole ed il ciliato Cryptocaryon irritans e il monogeneo Furnestinia echeneis nelle orate, sebbene sia stato possibile studiare anche il coinvolgimento di altri ectoparassiti, quali il monogeneo Sparicotyle chrysophrii ed il dinoflagellato Amyloodinium ocellatum, nel determinismo di alcuni episodi morbosi. Le osservazioni istopatologiche hanno permesso di caratterizzare le lesioni causate dagli ectoparassiti a diverse intensità d’infestazione. Per quanto concerne la ricerca di parassiti zoonosici, con particolare riferimento agli stadi larvali di nematodi Anisakidae del genere Anisakis, si sono condotti esami parassitologici a livello di cavità viscerale e di muscolo laterale in tutti i soggetti provenienti da allevamenti in gabbia (626 soggetti, di cui 441 spigole e 185 orate). Tutti i soggetti esaminati sono risultati negativi, indicando come il rischio di infestazione da larve di nematodi anisakidi possa essere considerato trascurabile in spigole ed orate allevate in gabbia, come già dimostrato per il salmone atlantico (EFSA, 2010).

Effect of eprinomectin treatment on milk yield and quality in dairy cows in South Tyrol, Italy

The effect of treatment with eprinomectin on milk yield, milk composition and somatic cell counts (SCCs) was studied in 105 dairy cows located on seven farms in South Tyrol, Italy. On each farm, half of the animals were treated with eprinomectin and the other half were used as an untreated control group. Three test day records per animal were obtained before treatment (days -117, -75 and -33) and another three test day records were obtained after treatment (days 22, 62 and 131). Test day records comprised milk yield, milk composition, SCC and days in milk. On the day of treatment, blood samples and faecal samples were taken for parasitological analysis. Cows with positive faecal egg counts yielded less milk. A significant effect of eprinomectin on milk yield was observed after treatment and was most pronounced on the second and the third test days after treatment (+1.90 kg [P=0.002] and +2.63 kg [P<0.001], respectively). Furthermore, a significant decrease in SCC was observed on the second test day after treatment.

Coprological study on intestinal helminths in Swiss dogs: temporal aspects of anthelminthic treatment

Coproscopic examination of 505 dogs originating from the western or central part of Switzerland revealed the presence (prevalence data) of the following helminthes: Toxocara canis (7.1%), hookworms (6.9%), Trichuris vulpis (5.5%), Toxascaris leonina (1.3%), Taeniidae (1.3%), Capillaria spp. (0.8%), and Diphyllobothrium latum (0.4%). Potential risk factors for infection were identified by a questionnaire: dogs from rural areas significantly more often had hookworms and taeniid eggs in their feces when compared to urban family dogs. Access to small rodents, offal, and carrion was identified as risk factor for hookworm and Taeniidae, while feeding of fresh and uncooked meat did not result in higher prevalences for these helminths. A group of 111 dogs was treated every 3 months with a combined medication of pyrantel embonate, praziquantel, and febantel, and fecal samples were collected for coproscopy in monthly intervals. Despite treatment, the yearly incidence of T. canis was 32%, while hookworms, T. vulpis, Capillaria spp., and Taeniidae reached incidences ranging from 11 to 22%. Fifty-seven percent of the 111 dogs had helminth eggs in their feces at least once during the 1-year study period. This finding implicates that an infection risk with potential zoonotic pathogens cannot be ruled out for the dog owner despite regular deworming four times a year.

Surface antigens and potential virulence factors from parasites detected by comparative genomics of perfect amino acid repeats

ABSTRACT: BACKGROUND: Many parasitic organisms, eukaryotes as well as bacteria, possess surface antigens with amino acid repeats. Making up the interface between host and pathogen such repetitive proteins may be virulence factors involved in immune evasion or cytoadherence. They find immunological applications in serodiagnostics and vaccine development. Here we use proteins which contain perfect repeats as a basis for comparative genomics between parasitic and free-living organisms. RESULTS: We have developed Reptile http://reptile.unibe.ch, a program for proteome-wide probabilistic description of perfect repeats in proteins. Parasite proteomes exhibited a large variance regarding the proportion of repeat-containing proteins. Interestingly, there was a good correlation between the percentage of highly repetitive proteins and mean protein length in parasite proteomes, but not at all in the proteomes of free-living eukaryotes. Reptile combined with programs for the prediction of transmembrane domains and GPI-anchoring resulted in an effective tool for in silico identification of potential surface antigens and virulence factors from parasites. CONCLUSION: Systemic surveys for perfect amino acid repeats allowed basic comparisons between free-living and parasitic organisms that were directly applicable to predict proteins of serological and parasitological importance. An on-line tool is available at http://genomics.unibe.ch/dora.

[Occurrence of Trichinella spp. in wild boar in Switzerland]

Trichinellosis is a worldwide occurring zoonosis caused by the intracellular nematode Trichinella spp. One of the main infection sources in Europe is raw or undercooked meat from wild boar. Trichinella britovi is prevalent in wild carnivores in Switzerland, thus a possible inclusion of wild boar in this wildlife cycle cannot be excluded. In order to assess the prevalence of Trichinella infection in wild boar, we tested 1,458 animals with both parasitological and serological methods. In none of the animals Trichinella-larvae could be recovered by the artificial digestion method (prevalence of larvae: 0 %; 95 % CI 0.0 - 0.3). Antibodies in meat juice were detected in 57 animals using a standardized E/S-Ag-ELISA. However, in the confirmatory westernblot, only 3 animals remained seropositive (seroprevalence: 0.2 %; 95 % CI 0.07 %-0.60 %). The occurrence of wild boar positive for anti-Trichinella-antibodies indicates that meat inspection for Trichinella-larvae in this species is important to prevent human infections.

Follow-up of 100 dogs with acute diarrhoea in a primary care practice.

This study aimed to examine the aetiology of acute diarrhoea and the relapse rate in 100 client-owned dogs presented to a first-opinion clinic. History, physical examination, faecal testing and owner questionnaire data were collected at initial presentation (T0) and at either the time of relapse or at a recheck performed within 3 months. All dogs received treatment according to their clinical signs. Of 96 dogs that completed the study, 37 (38.5%) relapsed during the study period, 21 (21.9%) relapsed within 3 months, and 16 others (16.6%) at 3 months to 1 year after initial examination. Dogs that had undergone a change in housing location within 1 month prior to presentation and dogs <1 year old were significantly more likely to have positive parasitological analyses (P=0.02 and P=0.001, respectively). Pica was a risk factor for relapse (P=0.0002).

Coprological analyses on apparently healthy Alpine ibex (Capra ibex ibex) from two Swiss colonies

To provide baseline parasitological data for health surveillance in free-ranging Alpine ibex (Capra ibex ibex), we assessed the endoparasite population and level of parasitism in apparently healthy ibex. Faecal samples from 148 ibex were collected between 2006 and 2008 in two different Swiss ibex colonies. They were analysed by coprology, including combined sedimentation/flotation method, sedimentation method, Baermann funnel technique and Ziehl-Neelsen staining. Gastrointestinal parasites and lungworms were identified in 100% and 81.8% of the examined animals, respectively. Highest prevalences were recorded for gastrointestinal strongylids other than Nematodirus/Marshallagia spp. (100%), Eimeria spp. (100%), Muellerius spp. (79.8%) and Nematodirus/Marshallagia spp. (79.0%). We report for the first time Cryptosporidium sp. in free-ranging Alpine ibex and Cystocaulus spp. in free-ranging ibex from Switzerland. On average, ibex were infected with 3.9 different parasites taxa (range: 1-8). Parasite prevalence and diversity varied significantly between sexes, study sites and seasons. Parasite egg output was low in 95.7% and moderate in 5.3% of the samples. Overall, the results indicate that Alpine ibex are widely infected with endoparasites and suggest that multiple infections are very common in apparently healthy populations. Furthermore, our data underline the potential influence of factors such as sex, study site and season on parasitological findings.

Evaluation of the diagnostic value of the ELISA tests developed by using EgHF, Em2 and EmII/3-10 antigens in the serological diagnosis of alveolar echinococcosis

Alveolar echinococcosis (AE), caused by larva stage of Echinococcus multilocularis, is one of the lethal parasitic diseases of man and a major public health problem in many countries in the northern hemisphere. When the living conditions and habits in Turkey were considered in terms of relation with the life cycle of the parasite, it was suggested that AE has been much more common than reported mainly from the Eastern Anatolia region of Turkey. Since in vitro serologic diagnosis tests with high specificity for AE have not been used in our country, most of the cases with liver lesions were misdiagnosed by radiological investigations as malignancies. The aim of this study was to evaluate the diagnostic value of the in-house ELISA methods developed by using three different antigens (EgHF, Em2, EmII/3-10) in the serological diagnosis of AE. The study samples included a total of 100 sera provided by Bern University Parasitology Institute where samples were obtained from patients with helminthiasis and all were confirmed by clinical, parasitological and/or histopathological means. Ten samples from each of the cases infected by E.multilocularis, E.granulosus, Taenia solium, Wuchereria bancrofti, Strongyloides stercolaris, Ascaris lumbricoides, Toxocara canis, Trichinella spiralis, Fasciola hepatica and Schistosoma haematobium were studied. In the study, EgHF (E.granulosus hydatid fluid) antigens were prepared in our laboratory from the liver cyst fluids of sheeps with cystic echinococcosis, however Em2 (E.multilocularis metacestode-purified laminated layer) and EmII/3-10 (E.multilocularis recombinant protoscolex tegument) antigens were provided by Bern University Parasitology Institute. Flat bottom ELISA plates were covered with EgHF, Em2 and EmII/3-10 antigens in the concentrations of 2.5 µg, 1 µg and 0.18 µg per well, respectively, and all sera were tested by EgHF-ELISA, Em2-ELISA and EmII/3-10-ELISA methods. For each tests, the samples which were reactive above the cut-off value (mean OD of negative controls+2 SD) were accepted as positive. The sensitivity of the ELISA tests performed with EgHF, Em2 and Em2II/3-10 antigens were estimated as 100%, 90% and 90%, respectively, whereas the specificity were 63%, 91% and 91%, respectively. When Em2-ELISA and EmII/3-10-ELISA tests were evaluated together, the specificity increased to 96%. Our data indicated that the highest sensitivity (100% with EgHF-ELISA) and specificity (96% with Em2-ELISA + EmII/3-10-ELISA) for the serodiagnosis of AE can be achieved by the combined use of the ELISA tests with three different antigens. It was concluded that the early and accurate diagnosis of AE in our country which is endemic for that disease, could be supported by the use of highly specific serological tests such as Em2-ELISA ve EmII/3-10-ELISA contributing radiological data.

Monitoreo de parásitos patógenos en afluentes agroindustriales

Las aguas residuales son potenciales portadoras de enteroparásitos patógenos para el hombre cuya dosis infectiva puede ser mínima y prolongado el tiempo de sobrevivencia de huevos y quistes en el ambiente. La reglamentación limita la presencia de huevos de helmintos y, hasta el año 2000, no proponía una técnica para su detección. Este trabajo se realizó para caracterizar parasitológicamente los efluentes procedentes de agroindustrias. La capacitación en la identificación microscópica demandó la confección de fichas que describen las características de huevos de helmintos. Se efectuaron ensayos con muestras de bodegas y aceiteras en los puntos detectados de contaminación. Los métodos fueron adaptados a las particularidades de los efluentes. Los resultados no señalaron presencia de helmintos; no obstante, se hallaron Cyclospora e Isospora, protozoarios que, por su alto poder infectivo, también constituyen un riesgo para el hombre. Sin embargo, no están contemplados por la legislación. Respecto a helmintos, es necesario un estudio comparativo de las técnicas de detección para efluentes agroindustriales, con el fin de establecer las que optimicen su recuperación. En cuanto a otras especies es preciso continuar investigando su presencia para evaluar la conveniencia de incorporarlas a la reglamentación.