Characterization of a novel cathepsin L-like protease from Taenia solium metacestodes for the immunodiagnosis of porcine cysticercosis.

Porcine cysticercosis is an endemic parasitic disease caused by infection with Taenia solium that is found predominantly in developing countries. In order to aid in the development of simple diagnostic approaches, identification and characterization of potential new antigens for immunodiagnostic purposes is desired. The cysteine protease family has previously been found to have important immunodiagnostic properties. These proteases are expressed as zymogens which contain a signal peptide, pro-peptide, and an active domain. Subsequent catalytic cleavage of the pro-peptide converts these zymogens into enzymes. With the use of bioinformatic tools we identified an active domain of a novel cathepsin L-like cysteine protease (TsolCL) in the T. solium genome. The TsolCL gene includes 705 nucleotides (nt) within a single intron and a 633 nt exonic sequence encoding an active protein of 211 amino acids. Sequence alignment and phylogenetic analysis suggest that the TsolCL gene is closely related to genes found in Echinoccocus granulosus and E. multiloculars. In addition, TsolCL was found to have a 61.9%-99.0% similarity to other cathepsin L proteins found in other helminths and mammals. We cloned, expressed, purified, and characterized the recombinant active TsolCL (27 kDa) using the baculovirus-insect cell expression system. TsolCL showed cysteine protease enzymatic activity with the capacity to hydrolyze the Z-Phe-Arg-AMC substrate as well as bovine serum albumin. However, TsolCL was not able to hydrolyze human immunoglobulin. In addition, TsolCL has cathepsin L conserved amino acid residues in the catalytic site (Gln8, Cys14, His159, Asn179 and Trp181) and the motif GCNGG. Using ELISA, TsolCL was able to distinguish circulating IgG antibodies between healthy animals and naturally infected pigs with cysticercosis, showing a moderate sensitivity of 83.33% (40/48; 95% CI: [69.8%-92.5 %]), and a specificity of 83.78% (31/37; 95% CI: [67.9%-93.8%]). In conclusion, a novel cathepsin L-like cysteine protease from a T. solium metacestode was expressed successfully in Baculovirus system and was evaluated as a candidate antigen to diagnose porcine cysticercosis using the ELISA immunoassay.

A novel enolase from Taenia solium metacestodes and its evaluation as an immunodiagnostic antigen for porcine cysticercosis.

Cysticercosis is a worldwide parasitic disease of humans and pigs principally caused by infection with the larvae of the pork tapeworm Taenia solium. Through the use of the recently-made-available T. solium genome, we identified a gene within a novel 1448 bp ORF that theoretically encodes for a 433 amino acid-long protein and predicted to be an α-enolase closely related to enolases of other flatworms. Additional bioinformatic analyses revealed a putative plasminogen-binding region on this protein, suggesting a potential role for this protein in pathogenesis. On this basis, we isolated the mRNA encoding for this presumptive enolase from T. solium metacestodes and reverse-transcribed it into cDNA before subsequently cloning and expressing it in both E. coli (rEnoTs) and insect cells (rEnoTsBac), in a 6xHis tagged manner. The molecular weights of these two recombinant proteins were ∼48 and ∼50 kDa, respectively, with the differences likely attributable to differential glycosylation. We used spectrophotometric assays to confirm the enolase nature of rEnoTs as well as to measure its enzymatic activity. The resulting estimates of specific activity (60.000 U/mg) and Km (0.091 mM) are quite similar to the catalytic characteristics of enolases of other flatworms. rEnoTs also exhibited high immunogenicity, eliciting a strong polyclonal antibody response in immunized rabbits. We subsequently employed rEnoTsBac for use in an ELISA aimed at discriminating between healthy pigs and those infected with T. solium. This diagnostic assay exhibited a sensitivity of 88.4% (95% CI, 74.92%-96.11%) and a specificity of 83.7% (95% CI: 69.29%-93.19%). In conclusión, this study reports on and enzymatically characterizes a novel enolase from T. solium metacestode, and shows a potential use as an immunodiagnostic for porcine cysticercosis.

Neurocysticercosis: A natural human model of epileptogenesis.

OBJECTIVE: To develop a better understanding of mechanisms of seizures and long-term epileptogenesis using neurocysticercosis. METHODS: A workshop was held bringing together experts in epilepsy and epileptogenesis and neurocysticercosis. RESULTS: Human neurocysticercosis and parallel animal models offer a unique opportunity to understand basic mechanisms of seizures. Inflammatory responses to degenerating forms and later-stage calcified parasite granulomas are associated with seizures and epilepsy. Other mechanisms may also be involved in epileptogenesis. SIGNIFICANCE: Naturally occurring brain infections with neurocysticercosis offer a unique opportunity to develop treatments for one of the world's most common causes of epilepsy and for the development of more general antiepileptogenic treatments. Key advantages stem from the time course in which an acute seizure heralds a start of the epileptogenic process, and radiographic changes of calcification and perilesional edema provide biomarkers of a chronic epileptic state.

Interspecies interactions and potential Influenza A virus risk in small swine farms in Peru.

BACKGROUND: The recent avian influenza epidemic in Asia and the H1N1 pandemic demonstrated that influenza A viruses pose a threat to global public health. The animal origins of the viruses confirmed the potential for interspecies transmission. Swine are hypothesized to be prime "mixing vessels" due to the dual receptivity of their trachea to human and avian strains. Additionally, avian and human influenza viruses have previously been isolated in swine. Therefore, understanding interspecies contact on smallholder swine farms and its potential role in the transmission of pathogens such as influenza virus is very important. METHODS: This qualitative study aimed to determine swine-associated interspecies contacts in two coastal areas of Peru. Direct observations were conducted at both small-scale confined and low-investment swine farms (n = 36) and in open areas where swine freely range during the day (n = 4). Interviews were also conducted with key stakeholders in swine farming. RESULTS: In both locations, the intermingling of swine and domestic birds was common. An unexpected contact with avian species was that swine were fed poultry mortality in 6/20 of the farms in Chancay. Human-swine contacts were common, with a higher frequency on the confined farms. Mixed farming of swine with chickens or ducks was observed in 36% of all farms. Human-avian interactions were less frequent overall. Use of adequate biosecurity and hygiene practices by farmers was suboptimal at both locations. CONCLUSIONS: Close human-animal interaction, frequent interspecies contacts and suboptimal biosecurity and hygiene practices pose significant risks of interspecies influenza virus transmission. Farmers in small-scale swine production systems constitute a high-risk population and need to be recognized as key in preventing interspecies pathogen transfer. A two-pronged prevention approach, which offers educational activities for swine farmers about sound hygiene and biosecurity practices and guidelines and education for poultry farmers about alternative approaches for processing poultry mortality, is recommended. Virological and serological surveillance for influenza viruses will also be critical for these human and animal populations.

Immunoinformatics prediction of linear epitopes from Taenia solium TSOL18.

Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18.

Utility of a protein fraction with cathepsin L-Like activity purified from cysticercus fluid of Taenia solium in the diagnosis of human cysticercosis.

Neurocysticercosis, an endemic parasitic disease in most developing countries, is caused by Taenia solium and compromises the human central nervous system. Cathepsin L-like proteases are secreted by several parasites including T. solium and constitute important antigens for immunodiagnostics. A protein fraction with cathepsin L-like activity was purified from the cysticercus fluid by size exclusion and ion exchange chromatography. Cathepsin L-like activity was measured fluorometrically by detecting the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC. The purified protein fraction included antigens of 53 and 25 kD that were tested in a Western immunoblot and in an enzyme-linked immunosorbent assay (ELISA) for detection of human cysticercosis. The sensitivity of the Western immunoblot was 96% for patients infected with multiple cysts and 78% for patients with a single cyst. Specificity was 98%. The sensitivity of the ELISA was 98% in patients with multiple cysts and 84% in patients with a single cyst. Specificity was 92.7%.

Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

Risk factors for canine echinococcosis in an endemic area of Peru.

An epidemiological study was conducted in a highland rural community in Peru to determine risk factors for canine echinococcosis caused by Echinococcus granulosus. Dogs were diagnosed using a coproantigen enzyme-linked immunosorbent assay (ELISA). Dog owners were interviewed prior to stool collection and asked for attitudes, practices and beliefs likely to be associated with local patterns of E. granulosus transmission. Univariate and multivariate analysis were used to determine odds ratios (OR) and their 95% confidence intervals (CI). The main risk factors found to be significantly associated with canine echinococcosis by univariate analysis were dog age (3-25 months) (OR, 5.14; CI, 1.7-15.7), female sex (OR, 4.3; CI, 1.4-13.3) and having been fed hydatid infected offal (OR, 2.9; 95% CI, 1.0-8.6). There was complete lack of knowledge about echinococcosis transmission. In addition to periodic dog treatment, control programs need to emphasize education of the human population to increase knowledge of parasite transmission and to change human practices associated with high rates of infection.

Screening for cystic echinococcosis in an endemic region of Peru using portable ultrasonography and the enzyme-linked immunoelectrotransfer blot (EITB) assay.

Cystic echinococcosis (CE) caused by the larval form of Echinococcus granulosus is a major public health problem in sheep-raising regions of the World. This study compared portable ultrasound with the enzyme-linked immunoelectrotransfer blot (EITB) assay as screening methods to estimate the prevalence of human CE in a remote village in the Peruvian Andes. Three hundred eighty-nine villagers were examined by portable ultrasound and blood samples were drawn by venipuncture. Sera were collected and tested for antibodies against CE using an EITB assay. Cystic lesions were classified based on their ultrasound morphologic characteristics. The prevalence of human CE using portable ultrasound and the EITB assay were 4.9% and 2.6%, respectively. Fifty-three percent of subjects with CE were EITB positive. Portable ultrasound was well received by the community, augmented CE detection and allowed a faster estimate of human infection than the EITB assay.

Field evaluation of a coproantigen enzyme-linked immunosorbent assay for diagnosis of canine echinococcosis in a rural Andean village in Peru.

One hundred and six dogs (61 males and 45 females) were examined for Echinococcus granulosus infection in a farming cooperative in the central highlands of Peru during November 1998. Canine echinococcosis was diagnosed using direct microscopic examinations of purged feces following arecoline purging and a coproantigen-detection enzyme-linked immunosorbent assay (ELISA) for E. granulosus. Mean age was 2 years with a range of 3 months to 9 years. The overall prevalence of canine echinococcosis using the ELISA test was 79% (84/106). Seventy-four dogs were successfully purged with arecoline. The frequency of canine echinococcosis was 82 (61/74) and 34% (25/74) by the coproantigen ELISA test and arecoline purging, respectively. The sensitivity and specificity of the coproantigen ELISA test was 88 and 95%, respectively. We found this assay to be especially advantageous in remote geographical areas. In future control programs against echinococcosis in Peru and other areas where E. granulosus is endemic the coproantigen ELISA should be used for the surveillance of the dog population.

Taenia solium oncosphere antigens induce immunity in pigs against experimental cysticercosis.

Immunity to Taenia solium infection was investigated using an experimental intramuscular oncosphere infection assay (IMOA) model in pigs. Three naturally infected pigs with cysticercosis were treated with oxfendazole (OFZ), a drug demonstrated to kill cysts in porcine muscle. These animals were then challenged with oncospheres but did not develop any cysts while three uninfected pigs that were similarly challenged, did develop intramuscular cysts. In another study, two groups of three pigs each were immunized with crude T. solium oncosphere and metacestode antigens, respectively, and tested with the IMOA. Immunization with crude oncosphere antigens (OAs) induced 100% protection, while metacestode antigens provided only partial protection. Immunoblots showed that pigs with complete immune protection to oncosphere intramuscular challenge had antibodies to two OAs at 31.3 and 22.5 kDa, respectively. Antibody to these two antigens was absent in pigs immunized with metacestodes or in uninfected control pigs. This study demonstrated the presence of two antigens that are unique to the oncosphere. Although, antibody to these two antigens is consistently present in pigs that are protected from an oncosphere intramuscular challenge their role in preventing infection by T. solium larval cysts is still hypothetical.

An objective procedure for ischemic area evaluation of the stroke intraluminal thread model in the mouse and rat.

Computer-assisted procedures are used to measure infarct areas in animal stroke models, but this approach usually follows the less objective manual tracing of the boundaries of the infarct. Building on previously reported methodology using scanned images of triphenyltetrazolium chloride (TTC)-stained rat brains in the intraluminal thread model, we developed an objective method to assess ischemic damage in both the mouse and rat brains. The unique addition to our approach is the use of sham-treated animals, which thereby permits the removal of normal brain white matter from the ipsilateral injured brain. All brain sections per animal were scanned simultaneously using a Microtek Scanmaker 4 flatbed scanner. Color segmentation on full color images of 2 mm coronal brain sections was performed. Using Image Pro Plus (4.0) and color segmentation, ischemic and normal white matter areas were measured in the green channel and the entire brain area in the red channel. The percent of unstained tissue was calculated for sham-treated animals and for those with cerebral ischemia. By subtracting the average unstained area of the sham-treated group from the average unstained area from the ischemic group, the ischemic area was calculated. This methodology was validated using mouse and rat permanent and transient, focal ischemia models and MK-801 in the permanent ischemia models. MK-801, dosed at 3 mg/kg i.p. prior to the injury, reduced the injury by 75% in the mouse and 44% in the rat permanent occlusion models. The benefits of this methodology include: objectivity of the analysis of the ischemic injury, use of readily available software so that costs can be contained and removal of normal subcortical white matter from the calculation. This method should allow more consistent evaluation of changes in the infarct size, therefore, resulting in reduced variability and higher productivity.

Quistes hepáticos no parasitarios: experiencia en 10 años / Nonparasitic hepatic cysts: a ten-year experience

Objetivo: describir la frecuencia de los quistes hepáticos no parasitarios y las características clínicas de los pacientes que los presentan. Antecedentes: los quistes hepáticos no parasitarios tienen una frecuencia baja comparados con el resto de la patología hepática y usualmente son asintomáticos. Sólo aquellos de gran tamaño o que se complican presentan manifestaciones clínicas. Material y método: se revisaron los expedientes con este diagnóstico de 1987 a 1997. Resultados: encontramos seis casos, cinco mujeres y un hombre, con edad promedio de 60.3 años, cuyo síntoma más común fue el dolor abdominal. Los signos más comunes fueron dolor a la palpación superficial y profunda, así como tumoración abdominal palpable. El diagnóstico se fundamentó además en ultrasonido, tomografía computarizada de abdomen y colangiografía retrógrada endoscópica. A dos pacientes que tenían comunicación a vía biliar se les realizó derivación interna y al resto drenaje y marsupialización. Conclusión: los quistes hepáticos no parasitarios son raros y evolucionan satisfactoriamente