Boletín chileno de parasitología
versión impresa ISSN 0365-9402
Bol. chil. parasitol. v.56 n.1-2 Santiago ene. 2001
Immunoblot analysis using antigen from Taenia crassiceps cysticerci
in the diagnosis of swine cysticercosis
1 Department of Veterinary Medicine, Federal University of Viçosa, 36571-000. Viçosa, MG, Brazil.
2) Faculty of Pharmaceutical Science, University of São Paulo, 05508-900. São Paulo, SP, Brazil.
3) Adolfo Lutz Institute, 01246-904. São Paulo, Brazil.
Se utilizó la técnica del inmunoblot para el diagnóstico de la cisticercosis porcina usando un antígeno total de cisticercos de Taenia crassiceps. Fueron analizados 13 sueros del cerdo con cisticercosis, 30 sueros controles negativos y ocho sueros del cerdo con hidatidosis, así como nueve del suino con macracantorincosis, 10 con ascaridiosis y ocho con pulmonía. El uso de este antígeno en el inmunoblot con suero de cerdos no se había publicado previamente. El inmunoblot fue padronizado por análisis de titulación en bloque mostrando 100.0% de sensibilidad y 96.7% de especificidad. Los péptidos específicos para la cisticercosis en orden de frecuencia fueron: 72-68 kD (100%), 16-15 kD (77%), 39-36 kD (62%), 18-17 kD (54%), 21 kD (31%), 14 kD (23%), 25-23 kD (8%), y 20-19 kD (8%). Reacción cruzada (72-68 y 18-17 kD) sólo se descubrió en una muestra (12.5%) de cerdo con hidatidosis. Debido a sus altas tasas de desempeño, el inmunoblot debe ser útil para confirmar el diagnóstico de cisticercosis porcina y es más eficaz que otras pruebas empleadas para este propósito, como examen de la lengua, examen anatomopatológico y ELISA.
Palabras clave (Key words): Cisticercosis porcina (swine cysticercosis); inmunoblot (immunoblot); Taenia crassiceps.
Swine cysticercosis is a disease caused by Taenia solium larvae (cysticerci). Its economic and sanitary effects n society are more important in developing countries in Asia, Africa and Latin America, where this disease should be considered a public health priority.
Swine infected with T. solium cysticerci play a fundamental role in the transmission and maintenance of human taeniasis and cysticercosis, with the consequent need for effective services of animal health and inspection of products o animal origin.
The high frecuency of human cysticercosis and the severity of the neurological manifestations occurring in this condition, with high morbidity and lethality, indicate the importance of the taeniasis-cysticercosis complex, justifying the application of effective measures of diagnosis and control of these illnesses in man and swine.
ELISA and immunoblot are particularly important among the immunological tests used for the diagnosis of swine cysticercosis (Gonzalez et al., 1990; Pinto et al., 2000). Investigating the prevalence of swine cysticercosis in an endemic area with four different tests, Gonzalez et. al. (1990) obtained the highest detection rate for the immunoblot (51.9%), followed by ELISA (37.7%), necropsy (31.2%), and tongue examination (23.4%). However, the cost of the immunoblot is more than three times the cost of ELISA (Díaz et al., 1992), although ist higher efficiency has been pointed out as its best advantage.
Due to the difficulty in obtaining larval antigens from T. solium cysticerci, Taenia crassiceps larval antigens have been used in immunologic tests for the diagnosis of swince (Biondi et al., 1996; Pinto et al., 2000) and human cysticercosis (Larralde et al., 1990; Vaz et al., 1997; Bueno et al., 2000).
Because of the incipient application of the immunoblot in the diagnosis of swine cysticercosis compared to human cysticercosis, the objective of the present study was to evaluate an immunoblot methodology using total antigen of T. crassiceps cysticerci (T-cra) and swine serum for the diagnosis of swine cysticercosis.
Based on anatomopathological swine examination, we analyzed by immunoblot 13 sera from swine with cysticercosis, 30 sera from negative controls and 35 sera from swine with no cysticercosis but with other diseases, i.e., hydatidosis (n=8), macracanthorhincosis (n=9), ascaridiosis (n=10), and Haemophilus or Mycoplasma pneumonia (n=8).
Crude antigen of T. crassiceps (T-cra) was submitted to SDS-PAGE at the concentration of 6 µg/mm, and was prepared by the method of Pinto et al. (2000). T-cra larvae were maintained in laboratory by intraperitoneal inoculation of female BALB/c mice and obtained as described by Vaz et al. (1997). The total cysts were dehydrated by lyophilization and then pulverized in a mortar. Saline was added to the material for homogenization with a Potter type blender in an ice bath. The mixture was then submitted to four ultrasound cycles, 30 sec, 1 mA, 20 Hz, followed by centrifugation at 16,800 g, 30 min, 4ºC.
The peptides, separated by SDS-PAGE on a 5-20%, gradient under reducing conditions using a discontinuous system, were transferred from the gel to 0.2 µm nitrocellulose membranes (Millipore Corp., USA) according to the methodology described by Towbin et al. (1979). The transfer was performed at room temperature over a period of one hour, at 50 mA and 17 V (Bio-Rad Laboratories, USA).
Strips of 3 to 4 mm width were obtained from the membrane blots and washed three times in saline solution (0.15 M NaCl) with 0.05% (v/v) Tween-20 and treated with blocking solution [5% skim milk in Tris-saline (10 mM Tris-hydroxymethylaminoethane and 0.15 M NaCl; pH 7.4)] For one hour with slow shaking.
Serum samples diluted in the 1:5 blocking solution were added to the strips and incubated for 14-18 hours at 4ºC with slow shaking. After six washes of 5 minutes each, the strips were incubated for one hour with peroxidase-labeled anti-swine IgG conjugate (A-5670). Sigma Chemical Co., USA) in diluting solution, and new washes were performed.
The reactive peptides were revealed with a chromogenic solution (5 mg diaminobenzidine, 1.5% H2O2 in PBS, pH 7.2) for about 10 minutes, and the strips were then washed with distilled water. The reactive bands were analyzed by scanning with a GS 700 densitometer and Rf and MW were calculated with the aid of the Molecular Analyst Program, version 1.4 (Bio-Rad Laboratories, USA).
The immunoblot was standardized by checkerboard titration using a positive and negative control serum. The best block was then confirmed by testing three positive (high, medium and low reactivity) and three negative control sera.
3. Analysis of the reactive peptides that discriminate between swine with cysticercosis and swine without cysticercosis.
we calculated the frequencey of the different peptides from the T.cra antingen reacting with the antibodies in the serum from the various swine groups. The location of the reactive bands on the nitrocellulose strip, as well as their physical appearance, particularly the color intensity, were considered in the interpretation of the reactivity and enumeration of the bands (Larralde, et al., 1989; Tsang et al., 1991).
The frequency of the reactive bands, the sensitivity, specificity, and positive and negative predictive values were calculated for each peptide in order to establish the criterion for serum differentiation as positive or negative for swine cysticercosis, or beter, for the definition of the specific peptides and for the establishment of the positive and negative criteria. After the criteria were established, the performance of the immunoblot was evaluated on the basis of the results for each group of swine.
The results of checkerborad titration showed that the best dilutions for the differentiation between positive and negative sera were 1:100 for serum and 1:1000 for the conjugate. This result was confirmed by the assay of additional samples and showed the importance o some peptides, mainly those of low molecular weight (< 25 kD), in discriminating between cysticercosis and non-cysticercosis. Among the specific peptides, particularly important were I(72-68 kD), which was reactive in the 13 (100%) positive samples, and Y(16-15), which was reactive in 10 (77%) positive sera. Despite this high sensitivity, the two peptides showed 7.6% and 1.5% rates of nonspecific reactions, respectively, with negative sera and with groups with other diseases. In contrast, the simultaneous reactivity (72-68 kD and 16-15 kD) showed 100% specificity and good sensitivity (10/13 = 77%).
The analysis of immunoblot data for all groups showed reactivity with 29 peptides ranging from 204 kD to 14 kD. This analysis was used to determine three criteria to differentiate sera of swine with cysticercosis from sera of swine that did not carry the disease (Table I).
Frequency of specific peptides and their performance rates in the immunoblot with Taenia crassiceps
antigens, according to cysticercosis (positive) and non-cysticercosis (negative and other diseases) groups.
|Serum groups||# Performance (%)|
* Specific peptides reactive with more than 50% of positive samples and with less than 10% of control group samples.
# S = sensitivity; SP= specificity; pv = predictive value.
Definition of specific peptide:
1- Absolutely specific peptide: a peptide that only reacted with positive control sera and did not react with negative sera or with the sera responsible for cross-reaction (100% specificity); this group consisted of the following five peptides: S(25-23 kD), T(22 kD), U(21 kD), V(20-19 kD), and Z(14 kD). Eleven (84.5%) positive sera reacted with at least one of these peptides: 3 (27%) wit one peptide, 4 (37%) with two peptides, 2 (18%) with three peptide, and 2 (18%) recognize the five absolutely specific peptides.
2- Relatively specific peptide: a peptide that reacted with more than 50% of the positive control sera and with less than 10% of the negative sera or with 10% of the sera from swine with other diseases (more than 50% sensitivity and more than 90% specificity); this group consisted of the following four peptides I(72-68 kD), O(39-36 kD), X(18-17 kD), and Y(16-15 kD).
3-Nonspecfic peptides: those that showed low sensitivity (<50%) and less than 90% specificity, including the remaining 20 peptides ranging from 204 to 74 kD, 66 to 40 kD and 35 to 26 kD.
Thus, the results shown in Table I led to the identification of nine specific peptides: I(72-68 kD), =(39-36 kD), S(25.23 kD), T(22 kD), U(21 kD), V(20-19 kD), X(18-17kD), Y(16-15kD), and Z(14kD).
Reactivity with two or more specific peptides was considered positive. Four positive control sera (30.8%) only reacted with two specific pwptides and the rest of them with three or more. The average number of bands that reacted with positive serum (13.5) was higher than the number of bands that reacted with the other two groups of sera (6.1 for the negative group and 6.7 for the group with other diseases); this difference was higher when only the specific peptides were considered, that is to say, 3.4 specific peptide for positive sera as opposed to 0.1 for negative sera and 0.2 for sera from the group with other diseases (Table I).
One serum (12.5%) of the eight with hydatidosis showed reactivity with relatively specific peptides (72-68 kD and 18-17kD) and with nonspecific (110-105 kD, 104-101 kD, 100-97 kD, 62-55 kD, and 50-45 kD) peptides. This serum corresponded to 2.9% of the 35 samples from animals with other diseases.
One negative control serum (3.3%) reacted with two specific peptides (76-68 and 39-36 kD) and other sera reacted with one of these peptides. The results presented in Table 100% sensitivity and 96.9% specificity, and 86.7% positive predictive value and 100% negative value for the immunoblot.
Reactivity of control sera in the immunoblot with Taenia crassiceps antigen
(other diseases: H hydatidosis, A ascariadiosis, M macracanthorhincosis, P pneumonia)
|* Specific peptides reactive with more than 50% positive samples and with less than 10% of control group samples.|
Several factors interfere with the performance of the immunoblot methodology, especially the need for trained personnel. The adequate and correct identification of reactive bands, as well as their enumeration depend on the researcher's experience, with special attention to their physical appearance and location on the nitrocellulose strips. Also, the mean value found in the analysis of the positive control samples should be considered, as well as the frequency of each reactive band for cysticercosis. (Larralde et. al., 1989; Tsang et. al., 1989). The efficiency of the immunoblot can also vary according to antigen lot, electrophoretic separation and transfer (Gershoni and Palade, 1983; Tsang et. al., 1983; Tsang et. al., 1989).
On the basis of the criterion used to define the peptides as "relatively specific" ("50% sensitivity and " 90% specificity), their reactivity profile was found to be similar to that of "absolutely specific" peptides, both of them being more discriminative than the non-specific peptides. The peptide group defined as specific (Table I) presented 98% specificity, with a highly specific performance in the immunoblot. The low sensitivity of some specific peptides (25-17 kD and 14 kD) seems to have been influenced by deficiencies in the transfer process of some of them to the nitrocellulose strips, which impaired some reactions with the antibodies of the positive control serum or rendered others too weak for visualization. This effect was also caused by the individual variation of the immune response, since the lot of swine positive controls was quite heterogeneous (Tsang et. al., 1991).
In a study on the immunologic response of pigs with cysticercosis from an endemic area and from a non-endemic area, Gonzalez et. al. (1994) detected differences among the animals. The reactivity of the immunoblot with positive sera of from the non-endemic area was less intense than that observed in the sera from highly infected animals. This difference was attributed previously to factors discussed by Tsang et. al. (1991) such as different alimentary habit, genetic susceptibility and different humoral responses.
It should be pointed out that the average occurrence of specific peptides was 0.1 for the negative control, 0.2 for the group with other diseases and 3.4 for the positive group, showing that the chosen criterior of positivity (at least 2 specific peptides) was highly rigorous and specific for the discrimination between cysticercosis and non-cysticercosis. According to Tsang et. al (1989), most of the samples from patients with cysticercosis recognized more than one of the specific bands, and 63.4% recognized at least six bands. All the swine experimentally infected with eggs of T. solium, by Tsang et. al. (1991) produced antibodies to more than one of the specific glycoproteins.
Of course, the criteria for defining positivity can vary according to the purpose of the research, i.e., whether the test should be more sensitive or more specific.
Peptides similar to these defined as specific in our investigation were considered important for the diagnosis of human cysticercosis using T. solium cysticerci: 26 and 8 kD (Gottstein et al., 1986); 105, 95, 70, 48 and 13 kD (Nascimento et al., 19995); 65, 45, 30, 24, 20 and 18 kD (Kaur et. al., 1996); 72-68 kD (Vaz et al., 1997); 50, 42-39, 24, 21, 18, 14 and 13 kD (Tsang et al., 1989; Diaz et al., 1992: Montenegro et al., 1994; Gonzalez et al., 1999); (Ito et al., 1999)
According to Tsang et al. (1989). low molecular weight glycopeptides from T. solium reacted with the majority (13 kD= 59%, 14 kD=48%, 18 kD=70%, 21 kD=82%, 24 kD=94%) of human sera from patients with cysticercosis. These glycopeptides were also reactive with sera from swine three weeks after experimental infection (Tsang et al., 1991). Other authors have been studying T. solium in the immunoblot in order to detect antibodies in sera from swine and have unanimously reported the immunodominance of low molecular weight glycoproteins from T. solium in swine cysticercosis: 23, 16, 11 and 8 kD (Pathak et al., 1994); 50, 42, 24, 18, 14 and 13 kD (Tsang et al., 1989; Aluja et al., 1996); 24, 19 and 13-12 kD (Evans et al., 1997).
Our results showed a similar pattern of reactivity for < 25 kD peptides from the T- cra antigen, indicating the importance of low molecular weight peptides also from the T. crassiceps cysticerci for the diagnosis and perhaps for studies on immunization and vaccines against swine cysticercosis.
The 50-45 kD peptide identified in the present study showed one of the lowest specificity rates (27.7%) and positive predictive values (14.5%), in agreement with other reports, (Gottstein et al., 1986; Larralde et al., 1989 Pathak et al., 1994).
In the present study, the serum from one swine with discrete lesions of predominantly cerebral location showed a weak reaction, in agreement with data reported by Pathak et. al. (1994).
We did not find any other published paper reporting the use of T. crassiceps antigen in immunoblot to study swine cysticercosis. The immunoblot performed here showed 96.9% specifity and 100.0% sensitivity.
Without affecting the good performance of the immunoblot, hydatidosis was the only disease that revealed antibodies with the potential capacity of reacting with specific peptides (72-68 e 18-17 kD) of the antigen used. Using purified T. solium glycoproteins, Tsang et al. (1989) did not report cross-reactions with human parasitoses including hydatidosis. Similarly to us, Montenegro et al. (1994) observed cross-reactions for peptides of crude T. solium antigen in human sera from hydatidosis, which disappeared when purified glycoproteins were used.
Considering the large number of specific peptides identified in the T-cra antigen, our results indicate the feasibility of its use, overcoming the difficulties in locating sources of T. solium (Vaz et al., 1997), and avoinding the antigen heterogeneity currently ocurring in the different lots of T. solium cysticerci and in the different laboratory lots of antigens (Larralde et al., 1990), characteristics that make it valuable for immunodiagnosis.
The use of T. crassiceps cysticerci will also be of value in studies that require large amounts of antigens, mainly those concerning purification, which is important for the improvement of the immunologic tests for the diagnosis of cysticercosis, and standardization of tests for application in epidemic surveillance of cysticercosis (Larralde et al., 1990).
The high frequencey of swine cysticercosis and of human cysticercosis and teniasis expected in certain countries justifies the application of efficient diagnostic methods that will permit effective measures of disease control both in terms of public health and animal health.
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Financial support: this work was partially supported by FAPESP 96/2235-8.
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