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 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.

Echinococcus granulosus: intraspecific genetic variation assessed by a DNA repetitive element.

A 186 bp Echinococcus granulosus-specific repetitive element, TREg, was used to assess genetic variation between strains. In G7 genotype (pig strain) it has the characteristics of a satellite DNA element with a copy number of 23000 per haploid genome. Analysis, by sequencing of TREg monomers, showed a great degree of identity within them. In the G1 genotype (common sheep strain) TREg-like repetitive elements were found in an interspersed distribution throughout the genome and in only 120 copies. The sequences of these monomers showed a great degree of variation between them and with TREg of G7 origin. The G6 genotype (camel strain) showed a pattern of distribution and copy number similar to the G7 genotype, and the G2 genotype (Tasmanian sheep strain) similar to the G1 genotype. Isolates from the G5 (cattle strain) and G4 (horse strain) genotypes also showed unique hybridization patterns in Southern blot experiments. The genomic plasticity of E. granulosus, which may have important consequences in the epidemiology and control of cystic hydatid disease is reflected in the results of this work.

Ultrasonographic diagnosis of ovine cystic echinococcosis.

The sanitary and economic impact of cystic echinococcosis is serious in those countries where it becomes endemic. Ultrasonography is one technique that may be used to diagnose this disease in endemic areas. In parasitized sheep, hydatid cysts appear sonographically as a round hypoechoic structure. Twenty two sheep destined for slaughter were studied sonographically and imaging findings compared to post-mortem findings. Three sheep with hydatid cysts were identified. Eighty additional sheep not destined for slaughter were also studied. Echinococcus granulosus cysts were detected in three animals. Forty sheep from a non-endemic area had no hepatic cysts. The in vivo sonographic study of sheep provides a useful screening tool for echinococcosis.

Immunodiagnosis of human fascioliasis by an enzyme-linked immunosorbent assay (ELISA) and a micro-ELISA.

Enzyme-linked immunosorbent assay (ELISA) and micro-ELISA were evaluated for their ability to detect anti-Fasciola hepatica antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis.

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.

Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts.

A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide/chloroform extraction and an adsorption to diatomaceous earth suspension. DNA suitable for polymerase chain reaction was obtained and used for parasite strain determination by mitochondrial cytochrome c oxidase I gene sequencing. Fertile and nonfertile cyst isolates from sheep, cattle, pigs, and humans were characterized. Hitherto, no direct parasite strain characterization has been made on nonfertile hydatid cysts, whereas here we report that nonfertile hydatid cysts were produced by sheep strain (G1 genotype) in sheep, cattle, and humans and by pig strain (G7 genotype) in pigs.

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.

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.

Genetic variation and epidemiology of Echinococcus granulosus in Argentina.

Polymerase chain reaction-ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes were used to characterize 33 Echinococcus granulosus isolates collected from different regions and hosts in Argentina, and to determine which genotypes occurred in humans with cystic hydatid disease. The results of the study demonstrated the presence of at least 4 distinct genotypes; the common sheep strain (G1) in sheep from Chubut Province and in humans from Río Negro Province, the Tasmanian sheep strain (G2) in sheep and 1 human from Tucumán Province, the pig strain (G7) in pigs from Santa Fe Province and the carnel strain (G6) in humans from Río Negro and Buenos Aires Provinces. The finding that pigs harboured the pig strain and the occurrence of the Tasmanian sheep strain has considerable implications for the implementation of hydatid control programmes due to the shorter maturation time of both strains in dogs compared with the common sheep strain. Furthermore, this is the first report of the presence of the G2 and G6 genotypes in humans which may also have important consequences for human health.

Echinococcus granulosus: cloning and characterization of a tandemly repeated DNA element.

A repetitive DNA element from the genome of the cestode Echinococcus granulosus has been cloned and sequenced. The 186-base-pair repeating units are arranged in direct tandem, probably clustered in the parasite genome. The estimated copy number of the repeat is 11,500 and represents between 2 and 3% of the parasite genome. The repetitive sequence is specific for Echinococcus since it does not cross-hybridize with either DNA of other cestode species or pig and dog DNA. The repetitive element is capable of detecting between 250 and 500 pg of E. granulosus DNA by dot blot assay.

Estrategias clasicas para el dignostico de criptosporidiosis en pacientes con SIDA y diarrea cronica / Classic estrategies for diagnosis of cryptosporidiosis in patients with AIDS and chronic diarrhea

El protozoario Cryptosporidium sp. ha sido reconocido crecientemente en asociación con enteritis grave en paceintes con el síndrome de immunodeficiencia. Los individuos estudiados comprendieron 84 adultos com SIDA y diarrea crónica. En este trabajo se describen 14 pacientes con infección intestinal causada por Cryptosporidium sp. La media del recuerdo de CD4 en estos pacientes fue (300 células/mm3 (en 7 de los 14). El examen de aspirados duodenales y heces incluyó preparaciones de muestras concentradas coloreadas con Kinjoun, Dimetilsulfóxido y Auramina. Se realizaron videoesofagogastroduodenoscopías (VEDA) para inspeccionar visualmente la mucosa y obtener biopsias. La VEDA reveló duodeno granular en 10 pacientes y jaspeado en uno de ellos. Las biopsias duodenales fueron coloreadas con hematoxilina-eosina, Giemsa y Azur II. Los acambios histológicos incluyeron atrofia (3/14), duodenitis (2/14) a ambos (3/14). La microscopía eletrónica de transmisión fue usada para la identificación de estadíos de desarrollo de Cryptosporidium sp.

[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.

Estudio morfologico de Enterocytozoon bieneusi en pacientes con SIDA y diarrhea cronica / Morphological study of Enterocytozoon bineousi in patients with AIDS and chronic diarrhea

Microsporidia are protozoan parasites responsible for significant gastrointestinal disease in patients infected with the human inmunodeficiency virus. We reporte 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 ó 50 cells/mm3. The spores were detected in smears from stool samples and duodenal aspirates stained with trichrome blue in all patiens. Light microscopy of semithin 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. Enterocytozzon bieneusi infection may be an important cause of diarrhea in patiens with AIDS in our country.

Detection of the microsporidian parasite Enterocytozoon bieneusi in specimens from patients with AIDS by PCR.

Microsporidia are protozoa parasites responsible for significant gastrointestinal disease in patients infected with human immunodeficiency virus. We evaluated a PCR assay of stool samples, duodenal aspirates, and biopsy specimens from patients with Enterocytozoon bieneusi infection. A 210-bp DNA fragment of the unique rRNA intergenic spacer could be amplified from all samples infected with E. bieneusi, but no amplification was seen by using DNA purified from samples with Septata intestinalis or other parasites and from negative control human cells. These results suggest that the PCR in stool samples may be a useful tool for the diagnosis of intestinal microsporidiosis in patients with AIDS.

Deteccion de portadores asintomaticos de quistes hidatidicos: aumento de la especificidad del ensayo immunoenzimatico

Una de las actividades que realizan los programas de control de hidatidosis causada por Echinococcus granulosus en la Republica Argentina es la busqueda de portadores asintomaticos de quistes hidatidicos entre la poblacion general que habita las areas de risco, mediante un ensayo inmunoenzimatico (EIE) con antigeno de liquido hidatidico total (EIE-ALHT) que selecciona los posibles portadores. En base a la experiencia recogida se ha observado que dependiendo de la prevalencia del area, entre el 10 por cento y el 30 por cento de las personas seleccionadas por el EIE con valores de densidad optica (DO * DO + 4S no presentan imagens compatibles con quistes hidatidicos. El proposito del estudio fue mejorar la especificidad de la prueba. Con ese fin, se evaluo la influencia de la modificacion de la oferta antigenica y el efecto de la absorcion de los anticuerpos anti-componentes sericos ovinos y anti-fosforilcolina de los sueros en estudios, 114 sueros no hidatidicos seleccionados por su alto nivel de reacciones cruzadas en EIE-ALHTy 118 sueros hidatidicos se estudiaron frente a 4 fracciones antigenicas de liquido hidatidico ovino. El EIE que empleo la fraccion antigenica S2B conjuntamente con la absorcion previa de los sueros (EIE-S2B/A) fue el sistema que mejor discrimino los sueros hidatidicos de los no hidatidicos

[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.