Seroepidemiological evidence for Taenia solium taeniasis/cysticercosis in three Venezuelan rural communities.

Taenia solium is the most common parasite infection of the brain, causing neurocysticercosis and typically found in rural communities with free-ranging pigs. Identification of transmission in rural areas is essential for its control. Risk factors and transmission of the parasite were evaluated in three rural Venezuelan communities (Valle del Rio and Potrero Largo, Cojedes state; and Palmarito, Portuguesa state) by a questionnaire (112 households) and coprological (492 samples) and serological (433 human and 230 porcine sera) analysis, respectively. Typical risk factors were found in all three communities: free-foraging pig husbandry, deficient sanitary conditions, high open defecation and ignorance of the parasite life cycle. Coprological examinations revealed a high level of soil-transmitted parasites. Importantly, two T. solium adult worm carriers were identified in each of the three communities. Anti-metacestode antibodies and the HP10 secreted metacestode glycoprotein were detected at significant levels in human and porcine sera in Valle del Rio, Potrero Largo and Palmarito. In conclusion, these communities may be considered to be endemic for taeniasis/cysticercosis, and the instigation of an appropriate control programme is recommended.

Transmission of porcine cysticercosis in the Portuguesa state of Venezuela.

The aim of this study was to assess transmission of Taenia solium cysticercosis in Palmarito Arriba, a small village in the rural area of the Portuguesa state of Venezuela, through (1) an evaluation of T. solium transmission risk factors present in the community and (2) serological detection of the secreted metacestode HP10 antigen (HP10 Ag) and of anti-metacestode antibodies in sera from rural pigs. Risk factors associated with transmission of cysticercosis were the following: 100% (23/23) of the households lacked piped water, 87.0% (20/23) of households lacked latrines, 88.0% (100/114) of inhabitants routinely defecated in the open/air, 19.05% (12/63) of the interviewed population had observed proglottids in their stools. More significantly, 9/13 householders breeding pigs reported seeing proglottids in their stools. Of the 25 pigs available for bleeding and serological testing, 64% (16/25) were free roaming and 36% (9/25) were "backyard" animals; 28% (7/25) were seropositive for both the HP10 Ag and antibody, 20.0% (5/25) were seropositive for HP10 Ag alone, and 36.0% (9/25) were seropositive for antibody alone. Given this clear evidence of endemic porcine cysticercosis, further studies are needed to assess and control the level of porcine and human taeniasis and cysticercosis in this and neighboring communities.

Selective B-cell expression of the MHV-68 latency-associated M2 protein regulates T-dependent antibody response and inhibits apoptosis upon viral infection.

To better understand the role of the M2 protein of the murine herpes virus strain 68 (MHV-68) in vivo, B-lymphocyte-restricted, M2-transgenic mice were constructed. The transgenic mice contained normal B-cell subpopulations in bone marrow, lymph nodes and spleen. After immunization with sheep red blood cells, spleens from M2-transgenic mice had increased germinal centres. Transgenic mice responded to the T-cell-dependent antigen keyhole limpet haemocyanin (KLH) with higher levels of secondary IgM and IgG2a antibodies than WT mice. Normal and M2-transgenic mice were infected with WT and M2 frame-shift mutant (M2FS) MHV-68 viruses. The pathogenesis of M2-transgenic mice infected with the M2-deficient mutant virus did not revert to that observed upon infection of normal mice with WT virus. However, the higher reactivation levels late after M2-transgenic mice were infected with WT virus reflected the importance of M2 as a target for the immune response, and thus with an impact on the establishment of latency. Finally, there was markedly less apoptosis in B-cells from M2-transgenic mice infected with either WT or M2FS mutant than from similarly infected WT mice, consistent with the published inhibitory influence of M2 on apoptosis in vitro. Thus, M2 provides a strategy to increase the pool of germinal centre B-cells through inhibition of apoptosis in the infected cell.

MHV-68 Open Reading Frame 20 is a nonessential gene delaying lung viral clearance.

Recently, it has been demonstrated that the MHV-68 ORF20-encoded gene product induces cell-cycle arrest at the G2/M phase, followed by apoptosis. To study the role of this conserved gene in vivo, two independent ORF20-deficient MHV-68 viruses and their revertants were constructed. As the replication in vitro of both mutants followed similar kinetics to that of the wild-type and revertant viruses, ORF20 is therefore a nonessential virus gene. No cell cycle arrest could be observed upon infection of cells with wild type MHV-68 or mutant viruses. In addition, no major differences were detected between mock- and virus-infected cells when protein and inactivation levels of the mitotic promoter factor cdc2/cyclinB were analyzed. Following intranasal infection, the recovery of mutant, revertant and wild-type viruses in the lungs was similar. With the ORF20-deficient viruses, however, there was a significant delay of four days in clearance of virus from the lungs. Surprisingly, the magnitude and cell population distribution in the exudates of the lung was essentially similar to mice infected with wild-type, revertant or ORF20-deleted viruses. Subsequent establishment of latency was normal for both mutants, demonstrating that ORF20 does not play a critical role in establishment of a persistent infection. These results indicate that while expression of ORF20 may impact on the pathogenicity of the infection, the observed induction of G2/M arrest in ORF20-expressing cells may not be the primary function of ORF20 in the context of viral infection.

A novel TLR3 inhibitor encoded by African swine fever virus (ASFV).

African swine fever virus (ASFV) encodes proteins that manipulate important host antiviral mechanisms. Bioinformatic analysis of the ASFV genome revealed ORF I329L, a gene without any previous functional characterization as a possible inhibitor of TLR signaling. We demonstrate that ORF I329L encodes a highly glycosylated protein expressed in the cell membrane and on its surface. I329L also inhibited dsRNA-stimulated activation of NFκB and IRF3, two key players in innate immunity. Consistent with this, expression of I329L protein also inhibited the activation of interferon-ß and CCL5. Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation. Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene. The demonstration of an ASFV host evasion molecule inhibiting TLR responses is consistent with the ability of this virus to infect vertebrate and invertebrate hosts, both of which deploy innate immunity controlled by conserved TLR systems.

The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex.

The conserved murine gammaherpesvirus 68 ORF20 has recently been demonstrated to induce G2 cell cycle arrest followed by apoptosis in human and mouse cells. Here, we demonstrate that its homologues UL24 in HSV-1, ORF20 in KSHV and UL76 in HCMV are also inducers of cell cycle arrest followed by apoptosis in both human and mouse cells. The mechanism of action is similar to that reported for MHV-68 ORF20, inactivating the mitotic complex cyclinB/Cdc2.

Expression, cellular localization and antibody responses of the African swine fever virus genes B602L and K205R.

Previously, we identified serological immunodeterminants of African swine fever virus (ASFV), including pK205R and pB602L, without homologues in the database. pK205R is expressed as a 33-kD protein from 4 h post-infection onward, initially diffusely distributed throughout cells, and subsequently in viral factories. pK205R was not found in purified virus. Both pK205R and pB602L are recognised by hyperimmune antisera from domestic pigs and bushpigs at late time points after infection, suggesting they may be useful diagnostically to distinguish animals persistently infected with virus.

Detection of HP10 antigen in serum for diagnosis and follow-up of subarachnoidal and intraventricular human neurocysticercosis.

INTRODUCTION: Neurocysticercosis (NC), a parasitic disease caused by Taenia solium, may be either asymptomatic or show a mild to severe clinical picture with intracranial hypertension. The most severe form of the disease is caused when viable cysticerci are localised in the ventricles or in subarachnoidal cisterns at the base of the skull. Detection of the secreted metacestode antigen HP10 in cerebrospinal fluid is a sensitive and specific method for the diagnosis of these severe NC cases. OBJECTIVE AND METHODS: To evaluate the validity of HP10 antigen detection ELISA when applied to serum, using paired serum and cerebrospinal fluid samples from 116 radiologically and clinically characterised NC patients. RESULTS: The HP10 antigen assay exhibited a similarly high sensitivity in identifying severe NC cases from sera (84.8%) and CSF (91.3%). In contrast, HP10 antigen was rarely detected in asymptomatic or mild NC cases (3 of 57). Importantly, the HP10 antigen assay applied to serum showed high specificity (94%) when used in 126 serum samples of non-NC subjects from an endemic community with a confirmed coproparasitological diagnosis of intestinal parasitic infections. Finally, the HP10 assay also proved to be of value in the follow-up of treated patients. CONCLUSION: This study confirms that detection of the metacestode HP10 antigen in serum is a useful tool for diagnosis and follow-up of patients with severe forms of NC treated with cysticidal drugs.

Molecular identification of Echinococcus granulosus genotypes (G1 and G7) isolated from pigs in Mexico.

With the aim of genotyping Echinococcus granulosus cysts found in Mexican livestock, we collected hydatid cysts from the livers and lungs of pigs in slaughterhouses in the state of Morelos, Central Region of Mexico. DNA was extracted from the parasites and examined by polymerase chain reaction (PCR) of rDNA internal transcribed spacer 1 (ITS1-PCR), Eg9-PCR, Eg16-PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP). In addition, fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) were sequenced. Two different genotypes of E. granulosus were unequivocally identified, the common sheep genotype, G1, and the common pig genotype, G7. The G1 genotype of E. granulosus has not been previously demonstrated in Mexico. Because of its recognized infectivity in humans, G1 genotype is a direct threat to human health and its presence in Mexico is consequently of immediate public health importance and epidemiological relevance.

Differential molecular identification of Taeniid spp. and Sarcocystis spp. cysts isolated from infected pigs and cattle.

In the present work, the species-specific identification of Taeniid spp. cysticerci and sarcocystis cysts isolated from infected pigs and cattle was achieved by PCR. In particular: (i) multiplex-PCR derived from HDP2 DNA fragment, specific for Taenia saginata/Taenia solium; (ii) PCRs and PCR-RFLPs of the rDNA internal transcribed spacers 1 and 2 (ITS1 and ITS2) for the differential diagnosis of taeniids; (iii) PCR derived from the 18S rRNA gene and sequencing, specific for Sarcoystis spp. The combined application of these three PCR protocols provided an unequivocally specific diagnosis of T. saginata, T. solium, T. hydatigena, Sarcocystis hominis and Sarcocystis suihominis, and may have practical application in the identification of calcified degenerating or morphologically dubious cysts, for example in the slaughter house situation or in human biopsy samples.

Parasitic diseases of the central nervous system: lessons for clinicians and policy makers.

Parasitic diseases of the central nervous system are associated with high mortality and morbidity, especially in resource-limited settings. The burden of these diseases is amplified as survivors are often left with neurologic sequelae affecting mobility, sensory organs, and cognitive functions, as well as seizures/epilepsy. These diseases inflict suffering by causing lifelong disabilities, reducing economic productivity, and causing social stigma. The complexity of parasitic life cycles and geographic specificities, as well as overlapping clinical manifestations in the host reflecting the diverse pathogenesis of parasites, can present diagnostic challenges. We herein provide an overview of these parasitic diseases and summarize clinical aspects, diagnosis, therapeutic strategies and recent milestones, and aspects related to prevention and control.

CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Circulating parasite antigen in patients with hydrocephalus secondary to neurocysticercosis.

End stages of neurocysticercosis include residual intraparenchymal brain calcifications and hydrocephalus. Although brain calcifications alone have a benign prognosis, hydrocephalus is frequently associated with chronic inflammation and intracranial hypertension, together with a protracted clinical evolution, and may lead to patient deaths. By using a monoclonal-based antigen detection enzyme-linked immunosorbent assay, we measured the levels of circulating parasite antigen in the sera of 56 patients with neurocysticercosis: 27 with calcifications only and 29 with hydrocephalus. The assay gave positive results in 14 of 29 patients with hydrocephalus but was consistently negative in patients with calcifications. Circulating parasite antigen in hydrocephalus secondary to neurocysticercosis indicates the presence of live parasites in these patients and thus a potential benefit from antiparasitic therapy.

Serological evidence for recent exposure to Taenia solium in Venezuelan Amerindians.

This study examined the seroprevalence and serum antibody isotype profile for Taenia solium cysticercosis in an Amerindian community in the Amazonas state of Venezuela. An antigen-trapping enzyme-linked immunosorbent assay (Ag-ELISA) was used to detect viable cysticercosis. Indirect ELISA (Ab-ELISA) and enzyme-linked immunoelectrotransfer blot (EITB) was performed by using antigens prepared from T. solium metacestodes to detect anti-parasite antibodies. The Ag-ELISA and Ab-ELISAs revealed 64.7% and 79.0% seropositivity, respectively, in the Amerindian population. Immunoglobulin (Ig) M was the predominant antibody class, suggesting recent infection. In comparison sera from, clinically defined, hospital neurocysticercosis cases revealed only 27% seropositivity by Ag-ELISA, compared with 86-92% seropositivity by Ab-ELISA, and IgG4 was the predominant antibody subclass detected. The EITB antigen recognition patterns of the hospitalized patients were very similar to that of the Amerindians, confirming exposure to the parasite. These results, combined with the predominance of IgM antibody responses and the marked detection of secreted products of viable parasites, strongly suggest that recent exposure to T. solium had occurred in the Amerindian population.

Detection of secreted cysticercal antigen: a useful tool in the diagnosis of inflammatory neurocysticercosis.

Neurocysticercosis is a common parasitic disease of the human central nervous system. It is particularly prevalent in developing countries, where it has a serious public health and economic impact. A major diagnostic problem with neurocysticercosis is its pleomorphic nature. Conventional diagnosis of neurocysticercosis still requires brain-computed tomography and/or magnetic resonance imaging, which are definitive but often prohibitively expensive and inaccessible in endemic areas. Herein, the monoclonal antibody HP10 antigen-trapping enzyme-linked immunosorbent assay, which has been used successfully to detect viable Taenia solium cysticercosis, was evaluated using cerebrospinal fluid (CSF) from Mexican neurocysticercosis patients with various defined pathologies. Sensitivity was higher in cases of inflammatory compared with non-inflammatory disease (94.1% vs. 33.3%) and in cases of multiple- compared with single-cyst cysticercosis (85% vs. 33.3%). Positivity was a strong indicator of active, inflammatory, multiple-cyst neurocysticercosis detecting 100% (15/15) of such cases. The overall specificity, as determined using CSF samples from patients with other neurological symptoms, was 97.7% (42/43). Since the assay only detects viable infection, it is of known value in the follow-up of treated patients to determine whether treatment has been successful. Thus, antigen detection may be of particular value in the assessment of symptomatic patients, who may potentially benefit from rapid treatment.

Taenia solium cDNA sequence encoding a putative immunodiagnostic antigen for human cysticercosis.

A T. solium metacestode cDNA library was prepared and antibody screened to obtain recombinant antigens, which could be used for the neurocysticercosis diagnosis. The F18 clone was selected and sequenced, and the full length cDNA characterised as well as the genomic structure from the gene. F18 is a single copy gene that spans approximately 6.1 kb and contains five exons and four introns. The F18 cDNA has a 690-nucleotide open reading frame that encodes a putative polypeptide of 229 amino acids with a predicted molecular mass of 26.06 x 10(3) M(r). The F18 recombinant protein was obtained and purified by affinity chromatography using pGEX system (G-F18) and pQE system (H-F18). The purified G-F18 fusion protein showed the best results when it was used in ELISA with sera from neurocysticercosis patients.

Taenia saginata derived synthetic peptides with potential for the diagnosis of bovine cysticercosis.

Immunity in Taeniids is predominantly antibody mediated and thus many serological immuno-determinants will have potential in both protection and diagnosis. The antigenicity of six peptides derived from four potentially protective molecules cloned from a Taenia saginata oncospheres cDNA library have been evaluated as targets for the specific diagnosis of bovine cysticercosis. The six peptides consist of: two peptides (HP6-2 and HP6-3) derived from the sequence of the 18 kDa surface/secreted oncospheral adhesion antigen identified by McAb-HP6, two peptides (Ts45W-1 and Ts45W-5) derived from the sequence of the T. saginata homologue of the T. ovis 45W protective gene family, one peptide (TS45S-10) derived from a T. saginata sequence with significant similarity to the T. ovis 45S protective antigen, and one peptide (TEG-1) derived from the sequence of the T. saginata homologue of Echinococcus spp. main surface protein. Longitudinal studies indicate that T. saginata infected cattle respond to all six peptides by 3-4 weeks post-infection and that the antibody levels remain high for at least 12 weeks post-infection. As protection against Taeniid parasites is predominantly antibody mediated, some of these six peptides may be of value as immuno-prophylactic tools and hence also in assays to determine resistance to infection with the parasite. For diagnosis, on the other hand, only three peptides (HP6-2, TEG-1 and Ts45S-10) performed with the necessary sensitivity and specificity to determine exposure to infection with T. saginata, and now merit an exhaustive evaluation prior to employment as routine diagnostic tools.

Virus manipulation of cell cycle.

Viruses depend on host cell resources for replication and access to those resources may be limited to a particular phase of the cell cycle. Thus manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment. For example, viruses capable of infecting nondividing cells induce S phase in order to activate the host DNA replication machinery and provide the nucleotide triphosphates necessary for viral DNA replication (Flemington in J Virol 75:4475-4481, 2001; Sullivan and Pipas in Microbiol Mol Biol Rev 66:179-202, 2002). Viruses have developed several strategies to subvert the cell cycle by association with cyclin and cyclin-dependent kinase complexes and molecules that regulate their activity. Viruses tend to act on cellular proteins involved in a network of interactions in a way that minimal protein-protein interactions lead to a major effect. The complex and interactive nature of intracellular signaling pathways controlling cell division affords many opportunities for virus manipulation strategies. Taking the maxim "Set a thief to catch a thief" as a counter strategy, however, provides us with the very same virus evasion strategies as "ready-made tools" for the development of novel antivirus therapeutics. The most obvious are attenuated virus vaccines with critical evasion genes deleted. Similarly, vaccines against viruses causing cancer are now being successfully developed. Finally, as viruses have been playing chess with our cell biology and immune responses for millions of years, the study of their evasion strategies will also undoubtedly reveal new control mechanisms and their corresponding cellular intracellular signaling pathways.

Recombinant antigen targets for serodiagnosis of African swine fever.

African swine fever (ASF) is an infectious and economically important disease of domestic pigs. There is no vaccine, and so reliable diagnosis is essential for control strategies. The performance of four recombinant ASF virus (ASFV) protein (pK205R, pB602L, p104R, and p54)-based enzyme-linked immunosorbent assays (ELISAs) was evaluated with European porcine field sera that had been established by Office International des Epizooties (OIE)-approved tests to be ASFV negative (n = 119) and ASFV positive (n = 80). The kappa values showed that there was almost perfect agreement between the results of the "gold standard" test (immunoblotting) and the results obtained by the p54-specific ELISA (kappa = 0.95; 95% confidence interval [CI], 0.90 to 0.99) and the pK205R-specific ELISA or the pB602L-specific ELISA (kappa = 0.92; 95% CI, 0.86 to 0.97). For the pA104R-specific ELISA, there was substantial to almost perfect agreement (kappa = 0.81; 95% CI, 0.72 to 0.89). Similar results were observed by the OIE-approved ELISA (kappa = 0.89; 95% CI, 0.82 to 0.95). Importantly, antibodies against these proteins were detectable early after infection of domestic pigs. Preliminary testing of 9 positive and 17 negative serum samples from pigs from West Africa showed identical results by the recombinant protein-based ELISA and the OIE-approved tests. In contrast, there was a high degree of specificity but a surprisingly a low level of sensitivity with 7 positive and 342 negative serum samples from pigs from East Africa. With poorly preserved sera, only the p104R-specific ELISA showed a significant reduction in sensitivity compared to that of the OIE-approved ELISA. Finally, these recombinant proteins also detected antibodies in the sera of the majority of infected warthogs. Thus, recombinant ASFV proteins p54, pB602L, and pK205R provide sensitive and specific targets for the detection of antibodies in European and West African domestic pigs and warthogs.

Neurocysticercosis: detection of Taenia solium DNA in human cerebrospinal fluid using a semi-nested PCR based on HDP2.

Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. This disease is usually diagnosed by radiology but the results are not always clear-cut and so immunological assays are often also used. A semi-nested PCR, based on the non-coding HDP2 sequence of T. saginata, has now been developed for detecting DNA from T. solium cysticerci and confirming NC. This PCR, which amplifies a 171-bp T. solium product, allowed the specific detection of just 174 attograms of T. solium DNA. The efficacy of the PCR was tested using cerebrospinal fluid (CSF) from neurological patients, including 46 confirmed Mexican cases of NC and 32 patients from non-endemic Spain. Eighteen of the confirmed cases [including 10 (71%) of the 14 with vesicular extraparenchymal cysticerci and four (17%) of the 24 with damaged cysticerci] and two (33%) of the six patients with 'uncertain' diagnosis (in whom a diagnosis of NC could not be established by radiological and immunological studies) were found PCR-positive. The 36 patients known to have neurological problems other than NC were found PCR-negative. The HDP2 PCR offers a new tool in the diagnosis of NC and in exploring the pathogenesis of this serious disease.