Epidemiological tools for effective surveillance of porcine cysticercosis in Africa

Porcine cysticercosis is widely distributed in developing countries. Many tools were developed for effective control of the tapeworm in endemic countries. Tongue examination, meat inspection and Ag-ELISA were widely used in epidemiological survey. Both tongue examination and meat inspection are highly specific but less sensitive. To improve performance of AgELISA, unambiguous test based on nanobodies was performed. Immunodiagnostic tests based on PCR, flows through assay (FTA), Surface Enhanced Laser Desorption–Ionization Time of Flight (SELDI-TOF) were also developed. Less data were reported using spatial statistical analysis hence multiples approaches were available for effective epidemiological survey of porcine cysticercosis.


Introduction
different areas in developing countries.In Africa, the tapeworm was identified in Western Africa, Eastern Taenia solium is a tapeworm responsible of Africa and Southern Africa.Meat inspection and serious public health problem in developing countries lingual examination are frequently used for the [1,2].This tapeworm is transmitted to human by absordiagnostic of the porcine cysticercosis in Africa.ption of T. solium eggs or when eating undercooked pork.In humans, the larval stage of T. solium is respon-Lingual examination: Lingual examination was widely sible for conditions named cysticercosis or neurocystiused in all the diagnostic studies dealing with the cercosis.Tongue examination and meat inspection are assessment of T. solium cysticercosis infection in the main methods used for identifying infected pigs.
Africa [10].To perform the test, the pig is firmly The specificity of the tests is assessed to be close to restrained in lateral recumbency and a hard wooden 100%; however the sensitivity is variable, depending stick is used to open the mouth and keep it open.Using very much on the degree of infection in the pig [3].
mutton cloth as an aid, the tongue is pulled out, Different techniques have been described to detect examined and palpated along its entire ventral aspect to antibodies and antigens of T. solium infections in man check for presence or absence of cysticerci.A pig found and pigs: enzyme linked immunosorbent assay (ELISA), to have one or more cyst(s) on the tongue is considered dipstick-ELISA, latex agglutination, immunoblot positive for T. solium cysticercosis.If carried out techniques, antigen detection based on sandwich correctly, the specificity of tongue palpation is close to enzyme-linked immunosorbent assay (Ag-ELISA) [3-100% [3,10].The sensitivity of the technique, is 9].Antigens detection based on Ag-ELISA was therehowever variable, depending very much on the degree fore effectively used to detect porcine and human of infection in the pig.A Bayesian approach was used cysticercosis.However, Ag-ELISA is not highly specific to estimate the performance of tongue inspection [3].and sensitive.Hence, other immunodiagnostics tests The performance indicators of this test were: 0.21 for and spatial statistical analyses of porcine cysticercosis sensitivity (se) and 1.00 for specificity (sp).None of the were developed in order to be used in epidemiological light infections and only about half of the heavy and survey.
moderate infections have been detected by tongue This paper aimed to review epidemiological tools inspection.Nevertheless, despite of the low sensitivity developed for the control of porcine cysticercosis.
of the lingual examination, this diagnostic test is useful in assessing of the spatial distribution of the tapeworm Macroscopic tools for porcine cysticercosis [11].
diagnostic Meat inspection: Post-mortem inspection for diagnostic Porcine cysticercosis is widely distributed among of T. solium cysticercosis was described [12,13].Meat inspection includes long and parallel incisions into the external masseter muscles on both sides of the face in an upward direction to severe completely the parotid gland below the ear.The abdominal and diaphragmatic several authors, Ag-ELISA test is useful for identifimuscles, the root of the tongue and the tongue of all cation of active infection [16,17].In contrast, cross reactions were detected with Taenia hydatigena [3,18].pigs should be incised and the blade of the tongue To improve the performance of Ag-ELISA in the viewed and palpated.The heart of all pigs will be viewed, detection of porcine cysticercosis, nanobodies derived palpated and opened up.A deep incision will be made into the septum.Incisions will also be made in the from camels was used to develop unambiguous triceps brachii muscle proximal to the elbow joint.
specific tests [19].Nanobodies owned remarkable Heavy infestation with Cysticercus cellulosae calls for characteristics such as being small, nonimmunogenic, carcass condemnation.In light or moderate infestation, very stable, highly soluble, and easy to produce in large the carcass may be conditionally approved pending quantities.Nanobodies technologies were developed heat or freezing treatment.Cysts that were encountered and used in order to control disease such as cancer.on incisional and intact surfaces were classified as [20].Nanobodies are antibody-derived single domain either viable (translucent, fluid-filled with invaginated proteins that contain the unique structural and whitish protoscolices visible) or degenerated (black, functional properties of only heavy chain antibodies sand-like or powdery contents) [14].The specificity of that naturally occur in camels [20].Nanobodies are meat inspection is estimated to be close to 100% [3, encoded by single genes and are efficiently produced in 15].But it is admitted that, meat inspection is less eukaryotic and prokaryotic hosts including bacteria sensitive [3].This test is unable to exclude T. solium and yeast [21].Because of their unique three-dimeninfected pork from the food chain since it failed to sional structure, nanobodies have access to cavities or detect infections in 61.3% of infected animals [15].
clefts on the surface of proteins [22,23].Nanobodies Thus, the method is imperfect for use in epidemioloare ideal tools to selectively stabilize specific conforgical surveys and cannot be applied to monitor control mational states of membrane proteins [24,25].Hence, programs of T. solium.Light infections go undetected nanobodies were cloned after immunization of in carcass inspection.Therefore, to control the parasite dromedaries with T. solium cysticercosis and phage effectively in endemic areas, more sensitive and procedure [19].The nanobodies that were obtained did specific diagnostic methods have been used, such as not cross react with Taenia hydatigena, Taenia saginata, immunodiagnostic tools [3].
Taenia crassiceps or Trichinella spiralis and were categorized into four epitopebinding groups.The ELISA for the detection of circulating T. solium glycoprotein 14kDa (Ts14), that was found, reacted antigens (Ag-ELISA) with antigen but the nanobodies also reacted with other Ag-ELISA (Antigens detection based on sandwich proteins of the same family.When tested with a sandwich enzyme-linked immunosorbent assay) test for the ELISA on cyst fluid, one particular nanobody detected detection of circulating T. solium antigens consists of its cognate serum antigens in a species-specific inhibicollection of about 5 ml of blood from the jugular vein tion ELISA. of large pigs or anterior vena cava of small pigs into On the other hand and in perspective to improve plain tubes with clot activator.After centrifugation, the Ag-ELISA response, the vesicular cyst antigen with 2serum from each tube is transferred into two labelled 0 Dimensional IgG immunoblot was used to test the cryovial tube aliquots for each pig and stored at -20 C reactivity of cysticercotic sera of pigs [26].The author until tested.The samples are first treated with trichloreported diversity among the proteins and antigens of roacetic acid (TCA) to break the antigen-antibody vesicular cyst and a diverse IgG antibodies response.complexes and then tested at a final dilution of 1:4.
Furthermore, using the random amplified polymorphic Briefly the sandwich assay consisted of coating the DNA markers (RAPDs) to assess the genetic plates with capturing monoclonal antibody (B158C11 variability among cysticerci infected pigs, authors A10), blocking, addition of TCA treated sera, after found genetically difference in cysticerci infected pigs which the second biotin labeled antibody (B60H8A4), [27].Therefore, a possible significant source of diversity streptavidin labeled peroxidase and ortho phenylin Taenia solium infectiveness and pathogenicity was enediamine (OPD) substrate are added consecutively.
suggested [26].Washings are carried out between the steps.The reaction is stopped using sulphuric acid and the plates nm.The cutoff is calculated using a modified Student t- The SELDI-TOF was used to identify biomarkers test programmed in MS Excel sheet, by comparing the of Taenia solium cysticercosis for viable cyst (active optical density of each serum sample with a series of disease) and degenerated cyst (inactive disease) [28].negative serum samples obtained from a pig without SELDI-TOF is a proteomic high-throughput technique, any history of cysticercosis in each study area at a which has been improved with the progression of probability level of P < 0.001.A serum sample is protein-chip systems.Three major components considered positive when the ratio (optical density of constitute the instrumentation of SELDI-TOF MS test sample/optical density cut-off) is > 1.0.The technology: protein chip arrays, a mass analyzer, and specificity and sensitivity of Ag-ELISA test were data analysis software.The protein chip arrays are the found to be 86.7% and 94.7%, respectively [3].For heart of the SELDI-TOF MS technology.Using chro-based on PCR protocol targeting mitochondrial cytomatographic surfaces to retain proteins and peptides on chrome oxidase (COI) gene as reported [46] is useful to ProteinChip arrays based on their physicochemical prove existence of the larval stages belonging to the Taenia genus [47].In contrast, PCR was used for the properties, the platform performs direct analysis via diagnostic of porcine cysticercosis after inspection of Time-Of-Flight-Mass Spectrography (TOF-MS).Peak the carcass [48].Nevertheless to perform this PCR patterns are formed via MS, which represent protein protocol, genomic DNA was extracted from T. solium expression profiles.The location and intensity of every cysticerci dissected from naturally infected cysticercosis peak in the pattern reflect the mass over charge ratio positive pigs.The oligonucleotide primers used were (m/z) and abundance of the corresponding protein TBR-3 (5'-GGC TTG TTT GAATGG TTT GAC G-3' [29,30] was reported [41][42][43] and twenty sources of variation flow through assay (FTA) method affecting SELDI-TOF process was found [44].SELDI-Flow-through assay (FTA) is a membrane based TOF MS is a high throughput technique that allows immunoassay.This assay is based on a principle of hundreds of samples to be screened for disease direct competitive ELISA [49,50].Antibody of the biomarker identification in a relatively short time [45].
tested molecule is coated on a membrane surface Otherwise, the system is manual, time consuming, and followed by an addition of tested molecule enzyme prone to human error.The cost of protein chip arrays conjugate.The molecule and its conjugate compete for seems to be high at $75/chip (8 spots).Using SELDIthe limited antibody binding sites.After dried step in an TOF, 13 specific biomarkers were found for the viable incubator and a washing step, the enzyme substrate is phenotype, 9 specific for the degenerated phenotype added and reacts with the tested molecule-coupled and 8 specific for either viable or degenerated cysts enzyme and color develops.A FTA for the serodiag-[28].Among biomarkers discovered in the experimental nostic of porcine cysticercosis using cyst fluid antigen sample, five biomarkers (clusterin, lecithin-cholesterol (CFA) and the whole cyst fluid antigen (WCA) has acyltransferase, vitronectin, and apolipoprotein A-I) been developed [51].The test consisted of coating the were identified to increase serum expression in pigs T. solium metacestode antigen on cellulose acetate infected with viable cyst.Haptoglobin was identified membranes in order to increase its capturing by the as biomarker that increase serum expression in animal antibodies in the serum sample.The bound antibodies infected with degenerated cysts.However, when tested are visualized by the addition of protein A colloidal biomarkers with field samples, only three biomarkers gold conjugate, which served as antigen-antibody were to specific to increase serum expression.
detecting reagent imparting pink color to the fluid antigen was prepared using Chung [52] method with modification.The proteins within the two A PCR protocol targeting mitochondrial antigens were estimated by using method of Lowry cytochrome oxidase (COI) gene in sera samples was with bovine plasma globulins as a standard [53].The used for the diagnosis of porcine cysticercosis [46].performance observed for FTA were better with CFA According to these authors, the use of PCR in serum for (96.0%sensitivity; 96.0% specificity) compared to the diagnosis of porcine cysticercosis has a high specificity WCA (92.0%sensitivity; 96.0% specificity) [51].and the assay did not showed cross-reactivity with When comparing result with the enzyme-linked immutoxoplasmosis and trichinellosis.However, this nosorbent assay, the sensitivity for both the antigens technique is also specific when used to test the other pig were 96 % while the specificities with CFA and WCA taeniids, mainly Taenia hydatigena, Taenia asiatica were 96 and 92 %, respectively.The authors found that [47] [55].In contrast comparing adjust for multiple testing and heterogeneous backvesicular fluid (VF) and a glycoprotein fraction (LLaground population densities, along with other confounding Gp fraction), purified from a whole parasite extracted variables.Kulldorff's spatial scan statistic is also by lentil lectin affinity chromatography, the vesicular applicable to both point and aggregated data, and has fluid ELISA showed 100% sensitivity and specificity been adapted to detect noncircular clusters [86,87,88].whereas the sensitivity and specificity was found lower Diggle's test and Stone's conditional test is used to with the LLa-Gp ELISA [56].Despite the variation on evaluate focal clustering [89,90] and to determine the sensitivity and specificity registered using CFA and whether risk declines from pre-specified point sources the WCA in Flow-through assay, this method of [76,85].When performed power evaluation of diseases diagnostic is rapid, easy-to-use [57].The method does clustering tests, spatial scan statistic has good power in not require any equipment and more importantly, any detecting local clusters [82].Tango's MEET is useful individual can perform this assay.However, since the for detection of urban clusters and Besag-Newell's test method is semi-quantitative, interpretation of results for detecting mixed clusters with a right choice of may be difficult when the tested molecule concenparameter.The Cuzick-Edwards' k-NN test was found tration of the test sample is close to the method cut-off to have a good power in detecting hot spot cluster but level.
the power of the test depends upon the right choice of

Spatial analysis tools for porcine cysticercosis
the parameter.With regard to the detection of global survey clusters, Tango's MEET has the highest power.Nevertheless, the spatial scan statistic performs diseases Disease analysis at geographical level would be clusters well, but not as well as Tango's MEET [82].beneficial for a better understanding of cause of However, Cuzick-Edwards' k-NN test is useful to diseases and actions to be promoted in the field of perform clusters with very small distance if a right epidemiological control.One of the techniques used to choice of parameter has been done.Statistical spatial reveal spatial distribution of diseases is clusters detection.
analysis approaches were seldom used to control In epidemiology, a cluster is a number of health events porcine cysticercosis [11,91].By using K functions to located close together in space and/or time [58].
assess general cluster of porcine cysticercosis and Identifying spatial and spatio-temporal clusters of SatScan to detect local cluster, authors found that the cases could help: (i) to generate new information for two approaches match well [11].In the district of further etiologic studies; (ii) to identify risk areas Matapalo (Peru), distance to nearest neighbor was used where to focus the surveillance and allocate the resources to detect hotspots surrounding taenia solium tapeworm (antibiotics, rapid diagnostic tests…); (iii) to develop carriers [92].The risk-adjusted nearest neighbour cost-efficient vaccination strategies [59].Spatial analysis hierarchical spatial clustering (Rnnh) was used to of diseases can be performed using approaches based detect clusters of porcine cysticercosis in a Rural Area on Bayesian geo-statistical, K-functions, Generalized of Eastern Zambia-A Community [93].SaTScan was Additive Models (GAM), Cuzick-Edwards test, Besag used to determine clusters of different indices of Taenia -Newell test [60,61] or SaTScan (Kulldorff M. and solium infection in Vellore district of Tamil Nadu State Information Management Services, http://www. in India [94].In western Kenya, the k-1 nearest satscan.org/).These aforementioned statistical spatial neighbours local convex hull method was used to analysis approaches were used to detect clusters of the determine the spatial ecology of free-ranging domestic following conditions: meningococcal meningitis, pigs [95].Campylobacter spp. in humans and in broiler flocks, Indeed the use of frequentist approaches above hension of cysticercosis distribution.describe, Bayesian approaches are also used to detect  It is recommended to combine immunodiagnostic test could be found to bind with T. solium proteins [19].
with spatial statistical tool for better control of the tapeworm.Nevertheless, both Ag-ELISA and EITB is useful for epidemiological survey because require access to Surface enhanced laser desorption-ionizationare read in a spectrophotometer at a wavelength of 492 time of flight (SELDI-TOF)

Development of immunodiagnostic test with membrane as a
dot.Performing the techinique, cyst polymerase chain reaction (PCR)

Contribution of tools in porcine cysticercosis diseases clusters. A Bayesian approach might be more control in developing countries
Furthermore, the use of this new collected on T. solium cysticercosis in Africa cannot approach could increase the detection of most active reflect the real situation of the diseases [106].However, sites for porcine cysticercosis transmission in endemic Ag-ELISA has the advantage to detect the presence of areas.Therefore, the detection of clusters become active infection and the level of the infective burden useful if authors assessed risks factors associated to[107].Nevertheless, to date the same sensitivity was diseases distribution in order to have better comprenot observed with the current available antigen capture

Table - 1
. Characteristics of porcine cysticercosis detection tests

Table - 2
. Characteristics of statistical tools use for diseases clusters detection Aggregated data, C: continuous data assays and the reported specificities are not yet close to control of porcine cysticercosis in zoonotic areas, it is levels required for mass screening of pigs in endemic interesting to develop diagnostic techniques with high areas [107,108].Performance of sandwich ELISA sensitivity and specificity, handy to use, rapid and cheap.could be improved with nanobodies if good epitope *less used, P: Point, D: dichotomous variable, Ag: