Bjorn Victor

Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis

Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.

Validation of meat inspection results for Taenia saginata cysticercosis by PCR-restriction fragment length polymorphism.

Bovine cysticercosis is a zoonosis caused by the larval stage (cysticercus) of the human tapeworm Taenia saginata. Infected cattle is an important food safety issue besides an economic concern. Humans get infected by eating raw or undercooked meat containing viable cysticerci. Visual meat inspection of bovines is the only public health measure implemented to control transmission to humans, but it lacks sensitivity and objectivity. It may underestimate the prevalence of the disease by a factor 3 to 10. Furthermore, the success of the method depends on the expertise of the meat inspector as well as on the stage of development of the cysticerci. The focus of this study was to develop and explore the usefulness of a PCR assay as an objective alternative to evaluate the meat inspectors visual inspection results. Hereto, a PCR was developed for the detection of T. saginata DNA in muscle lesions. Based on the laboratory classification of lesions, almost 97% of viable cysts were confirmed by PCR, while for dead cysts, the percentage was approximately 73%. Taken together, these data demonstrate the difficulties of visual meat inspection and their objective interpretation, emphasizing the need to improve current assays to strengthen the control of bovine cysticercosis.

Proteomic analysis of Taenia solium metacestode excretion-secretion proteins.

The metacestode larval stage of Taenia solium is the causal agent of a zoonotic disease called cysticercosis. The disease has an important impact on pork trade (due to porcine cysticercosis) and public health (due to human neurocysticercosis). In order to improve the current diagnostic tools and to get a better understanding of the interaction between T. solium metacestodes and their host, there is a need for more information about the proteins that are released by the parasite. In this study, we used protein sequences from different helminths, 1DE, reversed‐phase LC, and MS/MS to analyze the excretion–secretion proteins produced by T. solium metacestodes from infected pigs. This is the first report of the T. solium metacestode excretion–secretion proteome. We report 76 proteins including 27 already described T. solium proteins, 17 host proteins and 32 proteins likely to be of T. solium origin, but identified using sequences from other helminths.

Epidemiology and genetic diversity of Taenia asiatica: a systematic review.

Taenia asiatica has made a remarkable journey through the scientific literature of the past 50 years, starting with the paradoxical observation of high prevalences of T. saginata-like tapeworms in non-beef consuming populations, to the full description of its mitochondrial genome. Experimental studies conducted in the 1980s and 1990s have made it clear that the life cycle of T. asiatica is comparable to that of T. saginata, except for pigs being the preferential intermediate host and liver the preferential location of the cysts. Whether or not T. asiatica can cause human cysticercosis, as is the case for Taenia solium, remains unclear. Given the specific conditions needed to complete its life cycle, in particular the consumption of raw or poorly cooked pig liver, the transmission of T. asiatica shows an important ethno-geographical association. So far, T. asiatica has been identified in Taiwan, South Korea, Indonesia, the Philippines, Thailand, south-central China, Vietnam, Japan and Nepal. Especially this last observation indicates that its distribution is not restricted to South-East-Asia, as was thought so far. Indeed, the molecular tools developed over the last 20 years have made it increasingly possible to differentiate T. asiatica from other taeniids. Such tools also indicated that T. asiatica is related more closely to T. saginata than to T. solium, feeding the debate on its taxonomic status as a separate species versus a subspecies of T. saginata. Furthermore, the genetic diversity within T. asiatica appears to be very minimal, indicating that this parasite may be on the verge of extinction. However, recent studies have identified potential hybrids between T. asiatica and T. saginata, reopening the debate on the genetic diversity of T. asiatica and its status as a separate species.

Canine leishmaniasis in Algeria: True prevalence and diagnostic test characteristics in groups of dogs of different functional type

A Bayesian approach was used to assess the prevalence of Canine leishmaniasis and evaluate three serological diagnostic tests: indirect fluorescent antibody test (IFAT), direct agglutination test, and particle gel immuno-assay (PaGIA) for Canine leishmaniasis (CL) in Algiers. Four hundred and sixty-two dogs were involved in this study and divided in four groups according to their functional type: stray dogs, farm dogs, national guard dogs and pet dogs. The stray dog group showed the highest prevalence of leishmaniasis (11.7%), followed by the national guard dogs (9.7%) and the farm dogs (5.9%). IFAT was shown to be the most sensitive test in all groups. However, IFAT specificity was considerably lowered in the farm dog group: 65.2% versus 94.5% for the stray dogs. A considerable drop in PaGIA specificity was noted in the stray dogs group. The results of the current study demonstrate the variability of test characteristics in different situations and underline the danger of using standard values, without verifying their appropriateness for the specific purposes.

Seroprevalence of Zoonotic Parasites in Pigs Slaughtered in the Kathmandu Valley of Nepal

For several years, the demand for pork has been on the rise in Nepal. To assess the importance of pork as a carrier of zoonotic agents, we performed a cross-sectional study in the Kathmandu Valley of Nepal, in which we serologically determined the infection status of slaughtered pigs with regard to three of the most important parasites transmitted through pork consumption: Trichinella spp., Taenia solium cysticerci, and Toxoplasma gondii. From 2007 to 2010, 742 pigs were sampled at slaughter, of which 0.1% (95% confidence interval [CI] 0.0-0.7%) were found positive for Trichinella infection, 13.8% (95% credibility interval [CrI] 0.8-28.5%) for T. solium cysticercosis, and 11.7% (95% CI 5.2-17.5%) for Toxoplasma infection. Further monitoring of the related animal and human disease burden and strengthening of food safety protocols throughout the pork production chain are strongly recommended.

Epilepsy is not caused by cysticercosis in The Gambia.

Objective  To determine whether epilepsy is caused by Taenia solium cysticercosis in The Gambia.

Specific detection and identification of African trypanosomes in bovine peripheral blood by means of a PCR-ELISA assay

The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n=18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n=42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.

A Pipeline for Differential Proteomics in Unsequenced Species

Shotgun proteomics experiments often take the form of a differential analysis, where two or more samples are compared against each other. The objective is to identify proteins that are either unique to a specific sample or a set of samples (qualitative differential proteomics), or that are significantly differentially expressed in one or more samples (quantitative differential proteomics). However, the success depends on the availability of a reliable protein sequence database for each sample. To perform such an analysis in the absence of a database, we here propose a novel, generic pipeline comprising an adapted spectral similarity score derived from database search algorithms that compares samples at the spectrum level to detect unique spectra. We applied our pipeline to compare two parasitic tapeworms: Taenia solium and Taenia hydatigena, of which only the former poses a threat to humans. Furthermore, because the genome of T. solium recently became available, we were able to prove the effectiveness and reliability of our pipeline a posteriori.

Partially sequenced organisms, decoy searches and false discovery rates.

Tandem mass spectrometry is commonly used to identify peptides, typically by comparing their product ion spectra with those predicted from a protein sequence database and scoring these matches. The most reported quality metric for a set of peptide identifications is the false discovery rate (FDR), the fraction of expected false identifications in the set. This metric has so far only been used for completely sequenced organisms or known protein mixtures. We have investigated whether FDR estimations are also applicable in the case of partially sequenced organisms, where many high-quality spectra fail to identify the correct peptides because the latter are not present in the searched sequence database. Using real data from human plasma and simulated partial sequence databases derived from two complete human sequence databases with different levels of redundancy, we could demonstrate that the mixture model approach in PeptideProphet is robust for partial databases, particularly if used in combination with decoy sequences. We therefore recommend using this method when estimating the FDR and reporting peptide identifications from incompletely sequenced organisms.