Identification of excreted iron superoxide dismutase for the diagnosis of Phtytomonas

An excreted iron superoxide dismutase (FeSODe) of pI 3.6 with a molecular weight of 28-30 kDa was detected in the in vitro culture of Phytomonas isolated from Euphorbia characias (SODeCHA) and from Lycopersicon esculentum (SODeTOM), in Grace's medium without serum. These FeSODe excreted into the medium had immunogenic capacity: the positivity of the anti-SODeCHA serum persisted to a dilution of 1/30,000, and for the anti-SODeTOM to 1/10,000 by Western blot. In addition, cross reaction was detected between the anti-SODe serum of Phytomonas isolated from E. characias against SODeTOM, and the anti-SODe serum from L. esculentum with SODeCHA. This characteristic offers the possibility of its use to diagnose plant trypanosomatids. The validation of the test was confirmed by experimental inoculation of tomato fruits with Phytomonas isolated from L. esculentum. At 7, 10, 15, and 21 days post infection, it was possible to detect the presence of the parasites with the anti-SODe serum of Phytomonas isolated from L. esculentum at a dilution of 1/250. These serological results were confirmed by visualization of the parasites by optical microscopy. The data of this study confirm that the SOD is sufficient to identify a trypanosomatid isolated from plants as belonging to the genus Phytomonas.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Low susceptibility of Achatina fulica from Brazil to infection with Angiostrongylus costaricensis and A. cantonensis

Introduction of Achatina fulica in Brazil has led to serious concerns about its role as vector for metaIylid worms: AngioIylus costaricensis and A. cantonensis. Experimental infection with both parasites was performed to evaluate the potential risk for their transmission by the giant African snail. Groups of 5 animals, both wild and bred at captivity were exposed at different inocula: 1, 5, and 10 ´ 10³ L1 of A. costaricensis and A. cantonensis. In all groups, few snails got infected and parasitic burden was low. Two different ways of infection were tested: ingestion produced higher numbers of L3 than the inoculation through an artificial hole in the shell. We also report the parasitological examination of 6 batches of wild A. fulica from Florianópolis, state of Santa Catarina, Brazil: only 1 out of 244 animals were infected with metaIylid larvae. Taken together these data indicate that the giant African snail occurring in Southern Brazil is not a permissive host for both AngioIylus species and does not represent a significant risk for transmission of these parasites.

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64% of all infections, P. vivax for 28%, and P. malariae for 4%. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4% of total cases. Children under 10 years accounted for 58% of the cases; the risk for malaria in this age group was 77% higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42%), P. malariae (0.25%), and P. vivax-247 (0.1%). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.

Chagas disease: what is known and what is needed - A background article

Chagas disease began millions of years ago as an enzootic disease of wild animals and started to be transmitted to man accidentally in the form of an anthropozoonosis when man invaded wild ecotopes. Endemic Chagas disease became established as a zoonosis over the last 200-300 years through forest clearance for agriculture and livestock rearing and adaptation of triatomines to domestic environments and to man and domestic animals as a food source. It is estimated that 15 to 16 million people are infected with Trypanosoma cruzi in Latin America and 75 to 90 million people are exposed to infection. When T. cruzi is transmitted to man through the feces of triatomines, at bite sites or in mucosa, through blood transfusion or orally through contaminated food, it invades the bloodstream and lymphatic system and becomes established in the muscle and cardiac tissue, the digestive system and phagocytic cells. This causes inflammatory lesions and immune responses, particularly mediated by CD4+, CD8+, interleukin-2 (IL) and IL-4, with cell and neuron destruction and fibrosis, and leads to blockage of the cardiac conduction system, arrhythmia, cardiac insufficiency, aperistalsis, and dilatation of hollow viscera, particularly the esophagus and colon. T. cruzi may also be transmitted from mother to child across the placenta and through the birth canal, thus causing abortion, prematurity, and organic lesions in the fetus. In immunosuppressed individuals, T. cruzi infection may become reactivated such that it spreads as a severe disease causing diffuse myocarditis and lesions of the central nervous system. Chagas disease is characterized by an acute phase with or without symptoms, and with entry point signs (inoculation chagoma or Romaña's sign), fever, adenomegaly, hepatosplenomegaly, and evident parasitemia, and an indeterminate chronic phase (asymptomatic, with normal results from electrocardiogram and x-ray of the heart, esophagus, and colon) or with a cardiac, digestive or cardiac-digestive form. There is great regional variation in the morbidity due to Chagas disease, and severe cardiac or digestive forms may occur in 10 to 50% of the cases, or the indeterminate form in the other asymptomatic cases, but with positive serology. Several acute cases have been reported from Amazon region most of them by T. cruzi I, Z3, and a hybrid ZI/Z3. We conclude this article presenting the ten top Chagas disease needs for the near future.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

Anopheles darlingi bionomics and transmission of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae in Amerindian villages of the Upper-Maroni Amazonian forest, French Guiana

French Guiana is one of the areas in South America most affected by malaria and where the disease has become a serious public health problem. In spite of this situation, little recent entomological data are available from the main localities where the disease occurs, even though they are crucial for development of an effective vector control strategy. A longitudinal entomological survey was carried out from March 2000-February 2002 in three Amerindian villages, namely Twenké, Taluène and Cayodé, located in the Amazonian forest of the Upper-Maroni area, to assess anopheline mosquitoes and malaria transmission dynamics. Anopheles darlingi (Diptera: Culicidae) was the most abundant mosquito species caught during the study. This efficient American malaria vector was active the entire year, but showed an evident peak of abundance during the main rainfall season, from April-June, with an average human biting rate of 255.5 bites per person per night. Parity rates were homogeneous all year, indicating no significant seasonal variability in female survival rates. Estimated vectorial capacity indices were higher during the rainy season, even though the risk of transmission was present throughout the year (VCI > 1). A total of 14 An. darlingi were found infected with Plasmodium falciparum, Plasmodium vivax or Plasmodium malariae. The annual circumsporozoite indices were 0.15, 0.14 and 0.05, and the entomological inoculation rates were 22.8, 27.4 and 14.4 infected bites per person per year in Twenké, Taluène and Cayodé, respectively. An. darlingiwas endo-exophagic and rather exophilic in these localities. The species was collected throughout the night but was more aggressive between 21:30-03:30 h and after 05:30 h. Parity rates were homogeneous during the entire night. Impregnated hammock and/or bed nets, coupled with the use of mosquito repellents, as well as the early treatment of malarial cases, appear to be the most suitable tools for fighting malaria in these Amerindian villages since the spraying of residual insecticides is inefficient because of vector biology and the housing structure.

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France

Water is a vehicle for disseminating human and veterinary toxoplasmosis due to oocyst contamination. Several outbreaks of toxoplasmosis throughout the world have been related to contaminated drinking water. We have developed a method for the detection of Toxoplasma gondii oocysts in water and we propose a strategy for the detection of multiple waterborne parasites, including Cryptosporidium spp. and Giardia. Water samples were filtered to recover Toxoplasma oocysts and, after the detection of Cryptosporidium oocysts and Giardia cysts by immunofluorescence, as recommended by French norm procedure NF T 90-455, the samples were purified on a sucrose density gradient. Detection of Toxoplasma was based on PCR amplification and mouse inoculation to determine the presence and infectivity of recovered oocysts. After experimental seeding assays, we determined that the PCR assay was more sensitive than the bioassay. This strategy was then applied to 482 environmental water samples collected since 2001. We detected Toxoplasma DNA in 37 environmental samples (7.7%), including public drinking water; however, none of them were positive by bioassay. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method. Alternative methods can be used in conjunction with this one to determine the infectivity of parasites that were detected by molecular methods.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24%) were infected. The transmission rates of Toxoplasma gondii were 7%, 24% and 59% in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91% and 99.5%, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52% and 99%, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53% and 64%, respectively, and specificity values were 91% and 92%. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Growth, cysts and kinetics of Borrelia garinii (Spirochaetales: Spirochaetacea) in different culture media

The aim of the present paper was to evaluate cyst formation and growth parameters of Borrelia garinii in a range of media differing in formulation and cost. A qualitative assessment of morphology and motility of B. garinii was conducted. All media were prepared aseptically and used in test tubes or Petri dishes. For each medium, the initial spirochete concentration was standardized to 10³ spirochets/mL. The following culture media were suitable to grow B. garinii: Barbour-Stoenner-Kelly, brain heart infusion and PMR. Growth was minimal at six weeks post-inoculation and maximum spirochete density was observed between 9-12 weeks. Often, the cultures developed cysts of different sizes, isolated or in groups, with a spiraled portion of variable sizes, mainly in unfavorable culture media. Brazilian Lyme disease-like illness, also known as Baggio-Yoshinari syndrome (BYS), is a new and interesting emerging tick-borne disease, caused by Borrelia burgdorferi sensu lato spirochetes, only during its cystic forms. It has been assumed that the peculiar clinical and laboratory features of BYS are consequential to the absence of a human sucker Ixodes ricinus complex tick at risk areas in Brazil, supporting the concept that the borrelia phenotypic expression pattern is modified as it is transmitted through the host.

The benefits of using selenium in the treatment of Chagas disease: prevention of right ventricle chamber dilatation and reversion of Trypanosoma cruzi-induced acute and chronic cardiomyopathy in mice

Cardiac damage is a frequent manifestation of Chagas disease, which is caused by the parasite Trypanosoma cruzi. Selenium (Se) is an essential micronutrient, the deficiency of which has been implicated in the development of cardiomyopathy. Our group has previously demonstrated that Se supplementation prevents myocardial damage during acute T. cruzi infection in mice. In this study, we analyzed the effect of Se treatment in cases of T. cruzi infection using prevention and reversion schemes. In the Se prevention scheme, mice were given Se supplements (2 ppm) starting two weeks prior to inoculation with T. cruzi(Brazil strain) and continuing until 120 days post-infection (dpi). In the Se reversion scheme, mice were treated with Se (4 ppm) for 100 days, starting at 160 dpi. Dilatation of the right ventricle was observed in the infected control group at both phases of T. cruzi infection, but it was not observed in the infected group that received Se treatment. Surviving infected mice that were submitted to the Se reversion scheme presented normal P wave values and reduced inflammation of the pericardium. These data indicate that Se treatment prevents right ventricular chamber increase and thus can be proposed as an adjuvant therapy for cardiac alterations already established by T. cruziinfection.

Biomphalaria alexandrina snails as immunogens against Schistosoma mansoni infection in mice

Despite effective chemotherapy, schistosomiasis remains the second largest public health problem in the developing world. Currently, vaccination is the new strategy for schistosomiasis control. The presence of common antigenic fractions between Schistosoma mansoni and its intermediate host provides a source for the preparation of a proper vaccine. The objective of this paper is to evaluate the nucleoprotein extracted from either susceptible or resistant snails to protect against schistosomiasis. The vaccination schedule consisted of a subcutaneous injection of 50 µg protein of each antigen followed by another inoculation 15 days later. Analyses of marker enzymes for different cell organelles [succinate dehydrogenase, lactate dehydrogenase (LDH), glucose-6-phosphatase, acid phosphatase and 5'-nucleotidase] were carried out. Energetic parameters (ATP, ADP, AMP, phosphate potentials, inorganic phosphate, amino acids and LDH isoenzymes) were also investigated. The work was extended to record worm and ova counts, oogram determination in the liver and intestine and the histopathological pattern of the liver. The nucleoprotein of susceptible snails showed reduction in worm and ova counts by 70.96% and 51.31%, respectively, whereas the nucleoprotein of resistant snails showed reductions of 9.67% and 16.77%, respectively. In conclusion, we found that the nucleoprotein of susceptible snails was more effective in protecting against schistosomiasis.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite

The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection.