Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool for vaccination.

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

Field evaluation of an enzyme immunoassay for detection of asymptomatic patients in a hydatid control program.

In South America programs to control hydatidosis caused by Echinococcus granulosus include the active search for asymptomatic patients through population surveys for the detection of antibodies against arc 5 antigens using the double diffusion arc 5 test (DD5). Though simple to perform and highly specific, DD5 is not practical for population studies due to the time lapse between testing and receiving results. This work evaluates the application of an enzyme immunoassay to screen sera for subsequent processing using DD5. The efficiency of an enzyme immunoassay screening/DD5 confirmation scheme vs. DD5 alone was compared within the framework of a control program. A total of 5,839 sera from residents of endemic areas was processed and 47 hydatid patients were detected by both schemes. The proposed enzyme immunoassay identified all sera having antibody activity against arc 5 antigens detectable by DD5 and ruled out 95.3% of sera which tested by DD5 would have produced negative results.

Immunodiagnosis of human hydatid disease: applications and contributions to a control program in Argentina.

The sequential development of the hydatid immunodiagnostic activities of the control program of the Province of Neuquén, Argentina is described. Test results were used to obtain immunological confirmation of clinical cases and to detect asymptomatic cyst carriers amongst residents of rural endemic areas. The information was also valuable for improving the accuracy of prevalence estimates of human hydatidosis and the quality of surveillance data in different areas of the Province, characterized by varying degrees of environmental contamination by Echinococcus granulosus. In population groups examined by radiologic and immunologic methods, the latter detected more cases. When only immunodiagnostic surveys were carried out, mostly liver but also pulmonary hydatidosis cases were detected. This experience illustrates the advantages which may be obtained in endemic areas through the local application of hydatid immunodiagnosis based on arc 5 positivity.

Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase.

Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.

Characterisation of a novel interspersed Toxoplasma gondii DNA repeat with potential uses for PCR diagnosis and PCR-RFLP analysis.

A novel Toxoplasma gondii interspersed repeat element (TgIRE), present in most of the tachyzoite chromosomes, was characterised. Two regions on the TgIRE sequence showed high identity to two different T. gondii expressed sequence tag cDNAs of unknown function, which seems to be TgIRE pseudogenes. Two set of primers were designed, 2-2' and 2-3, that amplify products of 1.02 and 0.62 kb, respectively. T. gondii DNA from RH and Me49 strains was amplified with TgIRE 2-2' primers, and the respective 1.02 kb products were digested with several endonucleases. Different fragment patterns by gel electrophoresis were found only with MboI. Sensitivity analysis revealed that the set 2-3 was more sensitive than 2-2', detecting by gel visualisation the amount of DNA equivalent to 1 and 10 parasites, respectively.

Expression of a cDNA encoding a Toxoplasma gondii protein belonging to the heat-shock 90 family and analysis of its antigenicity.

A cDNA clone (Tgzy85d11.r1) obtained from the Toxoplasma Expressed Sequence Tag project was chosen due to its homology with proteins of the heat shock 90 family. The cDNA encodes 137 amino acids of the C-terminal portion of the Toxoplasma Hsp90 protein (TgHsp90). Serum samples obtained from orally infected BALB/c and C57BL/6 mice showed reactivity against a recombinant TgHsp90 (rTgHsp90) after 8 weeks postinfection. Isotype analysis showed an anti-rTgHsp90 IgG2a/IgG3 response in infected BALB/c and anti-rTgHsp90 IgG1/IgG2a/IgG2b response in infected C57BL/6 mice. Serum samples from individuals chronically and putative acutely infected with T. gondii showed a similar anti-rTgHsp90 IgG response. Our work identifies TgHsp90 as a novel parasite antigen that seems to elicit a higher relation of anti-TgHsp90/anti-T. gondii IgGs during chronic infection in comparison with the acute stage.

Urban outbreak of a Brucella melitensis infection in an Argentine family: clinical and diagnostic aspects.

An outbreak of Brucella melitensis in a family was studied. From the fourteen family members who ate unpasteurized goat cheese nine became ill. Patients included four females and five males of 8 to 75 years old. In seven of the patients the diagnosis was confirmed by positive blood culture for B. melitensis biovar 1. All the patients were analyzed by standard tube agglutination (STA) and standard tube agglutination with 2-mercaptoethanol (STA-2ME) tests at the time of diagnosis. In six of the patients, ELISA assays were used to assess the humoral immune anti-protein and anti-lipopolysaccharide (LPS) responses. Anti-LPS IgG antibodies were detected in all of the patients. Anti-proteins IgG antibodies were present at significant levels in all the studied patients including the STA-2ME negative ones.

Immunodiagnosis of pulmonary hydatid disease in a patient with negative radiologic and scintillographic findings.

Hydatid disease was diagnosed by the arc 5 double diffusion (DD5) test in a patient with concomitant pulmonary disease. The localization of the cysts could not be determined by radiologic and scintillographic studies of the lung and abdomen. The hydatid nature of fluid collected by pleural puncture required for the concomitant infection, was established by using the aspirate as antigen in the DD5 test and five small pulmonary hydatid cysts were found at surgery.

Seroepidemiology of human hydatidosis: use of dried blood samples on filter paper.

Programmes for the control of hydatidosis caused by Echinococcus granulosus in Argentina use an enzyme immunoassay (EIA) as a screening test for population surveys aimed at detecting asymptomatic patients. Persons thus selected are referred to health centres for the arc 5 double diffusion test and imaging techniques. One of the most costly procedures of these surveys is the collection of blood samples under field conditions; the possibility of collecting dry blood samples on filter paper was therefore investigated. In a survey of 497 rural inhabitants of an endemic area, the same number of hydatidosis cases (22) were identified by EIA using (i) serum samples and (ii) capillary blood samples obtained by finger prick and collected on filter paper. The latter system was both simpler and cheaper.

Canine echinococcosis: an alternative for surveillance epidemiology.

The essential activities for programmes of cystic echinococcosis control are the census of all dogs from the program and identification of parasitised animals. Currently, in South America evaluations and epidemiological surveillance are based on the administration of arecoline hydrobromide. This method has the disadvantage of increasing environmental pollution and risk for operators and owners of treated dogs. A genus-specific ELISA capture method has been employed for recently issued faeces and the confirmation of positive examination was performed by dog autopsies. Our work presents an alternative method based on collection of dry field-dispersed faeces, followed by serological diagnosis by Copro-ELISA and confirmation by Copro-Western blot. If Copro-ELISA were used to define positive samples of dry faeces, the Copro-Western blot assay would provide 70% sensitivity and 100% specificity. Global efficiency of the system using dry faeces would reach 76%, allowing epidemiological surveillance to be oriented to analysis of surface units instead of dog as measurement unit.

[Detection of asymptomatic carriers of hydatid cysts: specificity increase of the immunoenzyme assay]. / Detección de portadores asintomáticos de quistes hidatídicos: aumento de la especificidad del ensayo inmunoenzimático.

An enzymoimmunoassay (EIE) as a screening test to select potential asymptomatic cyst carriers among the general population of areas under risk is being used in programs for the control of hydatic diseases caused by Echinococcus granulosus in Argentina. The experience obtained up to date, applying this assay in population surveys, indicates that depending on the prevalence in the area 10% to 30% of the individuals selected did not show images compatible with hydatic cysts. The purpose of the present study was to improve the specificity of the test. To this purpose, the influence of the modification of the antigenic availability and the effect of the absorption from the serum samples of antibodies anti-normal ovine sera and anti-phosphorylcholine was evaluated. One hundred and fourteen non hydatic sera selected because of their high cross reactivity in EIE using the whole hydatid antigen (WHA) and 118 hydatid sera, were studied with four fractions of ovine hydatid cyst fluid. The EIE employing the S2B antigenic fraction with previous absorption of the sera (EIE-S2B/A) was the system that discriminated better hydatid sera from non hydatid sera with high levels of cross reactivity. The replacement of the EIE employing WHA by the EIE-S2B/A system, for the active search of asymptomatic cyst carriers in field conditions, is proposed. The four antigenic fractions were analyzed by double diffusion and SDS-PAGE. The S2B fraction revealed a high content of parasitic components of less than 30 Kd which probably includes antigen B and subunits or fragments of antigen 5.

[Community participation and appropriate technology in the early diagnosis of human hydatidosis]. / Participación comunitaria y tecnología apropiada en el diagnóstico precoz de la hidatidosis humana.

The basic strategy for development of hydatid struggle programs is in the actuality: Primary Attention of Health. In the present work and in this instance, it's arm a precocious detection system of hydatid disease, fixed in immunologic diagnostic by means of ELISA technical beginning with blood capillary samples, taken in filter paper by teachers and sanitary agents from official services of Rio Negro Province. 177 teachers and 45 sanitary agents were trained, correspondent to 25 schools, 3 lodging schools and 9 Hospitals all of them from rural area. 890 blood samples during the training were obtained. Lastly, the trained personal armed the system and they obtained 728 samples in the beginning of the Program. It hadn't statistical differences in the reactivity of both samples. The serological prevalence found was 1.32%. The activity displayed by teachers and sanitary agents permitted to detect 21 new cases it was the 20% of new cases diagnosed in this area in the period of work. The viability and the importance of the incorporation of non traditional effectors into the Hydatid Control Programs is discussed.

[Classic strategies for diagnosis of cryptosporidiosis in patients with AIDS and chronic diarrhea]. / Estrategias clásicas para el diagnóstico de criptosporidiosis en pacientes con SIDA y diarrea crónica.

Cryptosporidium sp., a protozoa organism, has been increasingly recognized in association with severe enteritis in patients with the Acquired Immunodeficiency Syndrome. The studied subjects included 84 adult patients with AIDS and chronic diarrhea. We describe 14 patients with intestinal infection caused by Cryptosporidium sp. The mean CD4 count in these patients was < or = 300 cells/mm3 (7 out of 14). Examination of duodenal aspirates and feces included dimethylsulfoxide, auramine and acid-fast preparation of concentrated samples. We carried out videoesophagogastroduodenoscopy (VEDA) to visually inspect the mucosa and obtain biopsy specimens. VEDA revealed granular duodenum in ten patients and jasper duodenum in one of them. Duodenal biopsy specimens were stained with hematoxylin-eosin, Giemsa and Azure II. Histologic changes included atrophy (3/14), duodenitis (2/14) or both (3/14). Transmission electron microscopy was used for the identification of developmental stages of Cryptosporidium sp.

[Morphological study of Enterocytozoon bineousi in patients with AIDS and chronic diarrhea]. / Estudio morfólogico de Enterocytozoon bieneusi en pacientes con SIDA y diarrhea crónica.

Microsporidia are protozoan parasites responsible for significant gastrointestinal disease in patients infected with the human immunodeficiency virus. We report the clinical features of three patients with chronic diarrhea and intestinal microsporidiosis caused by Enterocytozoon bieneusi. The average value for CD4 in these patients was < or = 50 cells/mm3. The spores were detected in smears from stool samples and duodenal aspirates stained with trichrome blue in all patients. Light microscopy of semi-thin plastic sections revealed parasites and spores in the enterocytes and were associated with villous atrophy (2 out of 3). Thin section-electron microscopy showed a variety of developmental stages of the microsporidio. Patients treated with Albendazole had an unsatisfactory clinical response to therapy. Enterocytozoon bieneusi infection may be an important cause of diarrhea in patients with AIDS in our country.

Usefulness of sero-surveillance for Trichinella infections in animal populations.

In this paper we evaluate serology as a tool to monitor Trichinella-free pig herds. Indoor, industrial-raised fattening pigs in the Netherlands are practically Trichinella-free, and were used as a negative reference cohort. A positive cohort was not available but we used sera from an endemic region in Argentina to model a plausible distribution of serological responses (as OD levels) in positive sera, employing the difference between the endemic sera and the negative Dutch sera. We describe a method for correcting for variation among ELISA plates using on-plate reference sera, and demonstrate how to apply these corrections to a collection of test sera from pig farms. The positive and negative reference distributions can be used to estimate fractions true and false positives, necessary for defining appropriate cutoffs to be used for classifying positive and negative animals. Based on this analysis, the serological test was shown to lack the predictive power required for its large scale deployment. The properties of the serological test were also compared to the conventional digestion assay, which is highly specific but considerably less sensitive.

Epidemiological survey of Trichinella infection in domestic, synanthropic and sylvatic animals from Argentina.

The presence of Trichinella larvae was investigated in 247 samples taken from domestic, synanthropic and sylvatic animals, collected during 1996 to 2005 in 12 endemic provinces of Trichinella infection in Argentina. Muscle larvae of Trichinella from 65 infected animals were identified at the species level by single larva nested polymerase chain reaction (PCR) technique based on the variability within the expansion segment V (ESV) region of the ribosomal DNA. Trichinella infections were found in 97 of 164 pigs, 38 of 56 pork products, two domestic dogs, one domestic cat, 7 of 11 armadillos and 3 of 9 synanthropic rats. All Trichinella isolates were identified as Trichinella spiralis by nested PCR. These findings add new data on the epidemiology of trichinellosis and should be considered when implementing new strategies to control this zoonosis.

High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

Immunodiagnosis of abdominal hydatid disease. Difficulties in the radiological and scintillographic localization of cysts.

A diagnosis of hydatid disease was established by the arc 5 double diffusion (DD5) test in two persons who had no symptoms, of whom one had had prior surgery for this parasitic infection. The location of some cysts which were removed surgically from these patients could not be determined in preoperative radiological and scintillographic studies. Postoperative serological monitoring by DD5 showed that one of these patients, though without symptoms, was harbouring an additional cyst. Abdominal x-rays showed no abnormalities, a partially calcified 2.7 cm cyst was detected in the right lobe of the liver by computerized axial tomography.