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Research Article
 

Diagnosis of Toxoplasmosis Using Affinity Purified Fraction of Tachyzoites Local Isolate



Soad E. Hassan, Nagwa I. Toaleb, Raafat M. Shaapan, Eman Hussien Abdel-Rahman and Ehab Kotb Elmahallawy
 
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ABSTRACT

To increase diagnostic potency of antigen, its immunogenic component must be isolated. Immuno-affinity column chromatography was adopted to purify immunogenic fraction of Toxoplasma gondii tachyzoites of chicken local isolate. The isolated fraction was characterized by SDS-PAGE, where it exhibited 2 bands of molecular weights 116 and 83 kDa compared with 8 bands of molecular weights ranged from 116-30 kDa in crude antigen. The isolated fraction proved high diagnostic activity compared with crude antigen by ELISA using two fold serial dilutions of infected human and chicken sera. The fraction detected antibodies in 366 out of 406 (90.1%) examined free range chicken serum samples. Also, it proved success in the detection of Toxoplasma antibodies in 30 serum samples of patient infected with T. gondii. The fraction proved 100% sensitivity and 84.4% specificity. Its Positive Predictive Value (PPV), Negative Predictive Value (NPV) and diagnostic accuracy were 85.7, 100 and 91.9%, respectively. In conclusion, the present study introduced novel T. gondii chicken tachyzoites partially purified fraction proved high diagnostic activity of chicken and human toxoplasmosis.

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Soad E. Hassan, Nagwa I. Toaleb, Raafat M. Shaapan, Eman Hussien Abdel-Rahman and Ehab Kotb Elmahallawy, 2016. Diagnosis of Toxoplasmosis Using Affinity Purified Fraction of Tachyzoites Local Isolate. Research Journal of Parasitology, 11: 13-19.

DOI: 10.3923/jp.2016.13.19

URL: https://scialert.net/abstract/?doi=jp.2016.13.19
 
Received: January 18, 2016; Accepted: February 15, 2016; Published: March 15, 2016



INTRODUCTION

Toxoplasmosis caused by Toxoplasma gondii is one of the most common parasitic infections of humans and other warm blooded animals. The cost of illness in the US caused by Toxoplasma has been estimated to be nearly US $3 billion annually (Batz et al., 2012; Hoffmann et al., 2012). Recent publications have linked suicide and schizophrenia to Toxoplasma infection (Torrey et al., 2012). Moreover, Mead et al. (1999) suggested that T. gondii is one of three pathogens (together with Salmonella and Listeria), which account for >75% of all deaths due to food borne disease in the USA.

Free rang-chicken is one of the major food sources for human. Therefore, it can be considered as a potential source of human toxoplasmosis (Wang et al., 2015). The rate of chicken toxoplasmosis was ranged from 30-50% (Zhu et al., 2008; Zhao et al., 2012), 78.12% in free rang chicken (Hassan et al., 2015) and reached to 100% in backyard chickens (Dubey, 2010a; Dubey et al., 2010). But chickens always display chronic infection without apparent clinical signs to toxoplasmosis (Zou et al., 2011). In addition, complicating matters is the fact that the number of T. gondii organisms in meat from naturally infected food animals are very low, making the parasite difficult and expensive to detect by direct methods. It is estimated that as few as one tissue cyst may be present in 100 g of meat (Dubey, 2010b). Moreover, there is no predilection site for Toxoplasma in meat animals, virtually all edible portions of an animal can harbor viable T. gondii tissue cysts and tissue cysts can remain viable in food animals for years (Dubey et al., 1986).

So, serological diagnosis considered as precise method for toxoplasmosis. On the other hand, infected chicken with toxoplasmosis play an important role in epidemiology of human toxoplasmosis (Dubey et al., 2004; Aboelhadid et al., 2013). Consequently, the aim of this study was to isolate immunodiagnostic fraction of chicken isolate Toxoplasma gondii tachyzoites antigen using affinity column chromatography and using it in diagnosis of chicken and human toxoplasmosis.

MATERIALS AND METHODS

Toxoplasma gondii chicken local strain: The strain was isolated from pooled tissues of heart, brain and pectoral muscles of slaughtered chicken at different regions in Egypt. The meat and tissue samples were digested using pepsin digestive solution as described by Sharma and Dubey (1981). Virulent local strain of T. gondii was obtained by bioassay of suspected infected chicken tissues in mice according the method of Dubey et al. (2010) and the obtained isolate was confirmed as T. gondii by polymerase chain reaction.

Antigen preparation: Soluble crude antigen was prepared from chicken tachyzoites local isolate according to Waltman et al. (1984), briefly, tachyzoites were repeatedly freezing and thawing to rupture the parasite wall, sonicated and centrifuged at 12,000 rpm for 45 min at 4°C. The supernatant was collected and its protein content was determined by the method of Lowry et al. (1951) and antigen was aliquoted and stored at -20°C until use.

Samples collection
Chicken serum samples:
Four hundred and six random chicken blood samples were collected from different localities in Egypt. Also, positive blood samples, which proved to be strongly positive by ELISA were selected and designated. Serum samples were obtained by centrifugation of coagulated blood, labeled in serial numbers and stored at -20°C until use.

Human serum samples: Sixty two human blood samples were investigated serologically by ELISA for detection of T. gondii IgG antibodies and parasitologically for other parasitic infections. According to these examinations, human sera were classified into 3 groups: Group (I) included 30 patients were positive for T. gondii IgG serologically and negative for other parasitic infections parasitologically. Group (II) included 21 patients were negative for toxoplasmosis and positive for other parasitic infections; 10 schistosomiasis mansoni, 5 fascioliasis, 3 amoebiasis and 3 hydatidosis. Group (III) included 11 individuals negative for toxoplasmosis and other parasitic infections used as control negative.

Purification of chicken T. gondii tachyzoites local strain antigen: Sepharose 4B affinity column chromatography was adopted for purification of chicken tachyzoites crude antigen. In brief, chicken T. gondii positive sera were dialyzed for three days against 0.1 M NaHCO3 buffer pH 8.3 containing 0.5 M NaCl and 0.02% NaN3 and coupled to cyanogen bromide-sepharose 4B (CNBr-Sepharose 4B) swollen beads (Sigma-Aldrich, USA) by strictly using the manufacturer instructions. Bound fraction was eluted with 50 mM glycine, pH 2.3 containing 500 mM NaCl pH 2.3 (Ahn et al., 1997). Protein concentrations of unbound and purified antigens were assayed by the method of Lowry et al. (1951).

SDS-PAGE: Crude chicken T. gondii tachyzoites antigen and its isolated fraction were separately mixed with sample buffer containing 2-mercaptoethanol before loading to the slab gel using 10% SDS-PAGE (Laemmli, 1970). After separation, gel was fixed in 50% methanol and stained with silver stain according to Wray et al. (1981). Relative molecular weights of bands were calculated using molecular weight marker.

ELISA: The diagnostic potency of crude antigen and the isolated fraction was assessed by indirect ELISA and two fold serial dilutions of infected chicken and human sera. The most potent antigen was adopted to detect toxoplasmosis infection in collected chicken and human serum samples. Antigen specificity was assessed against human infected serum samples with schistosomiasis mansoni, fascioliasis, amoebiasis and hydatidosis. The ELISA was performed, antigens concentrations, sera and conjugate dilutions were determined by checkerboard titration (Santiago and Hillyer, 1988) and the cut off value was calculated were determined according method of Shaapan et al. (2008).

Statistics: Statistical analysis was performed with unpaired student’s t-test using computer software package GraphPad Prism 5.02 version to compare groups. Sensitivity, specificity, diagnostic accuracy, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of isolated fraction were calculated according to Gonzalez-Sapienza et al. (2000).

RESULTS

Electrophoretic profile of antigens: The isolated fraction was resolved into two bands of 116 and 83 kDa in lane 2 (Fig. 1) compared with 8 bands associated with crude extract (116, 87, 83, 66, 55, 40, 36 and 30 kDa in lane 1 (Fig. 1).

Diagnostic potency of crude extract and isolated fraction: Isolated fraction proved higher diagnostic potential compared with crude extract in detection of chicken and human toxoplasmosis (Fig. 2a and b), respectively. Activity of isolated fraction was still potent, where it detected Toxoplasma IgG at dilution (1:4096).

Diagnostic potential of isolated fraction: Based on checker board titration results for ELISA, the optimum concentration of antigen was 20 μg mL–1, while the dilution of antibodies was 1:100 and the dilution of conjugate was 1:1000. The isolated fraction can detect Toxoplasma antibodies in 366 out of 406 (90.1%) of examined chicken serum samples (Fig. 3).

Fig. 1:
Electrophoretic profile of the isolated fraction (lane 2) compared with crude extract of T. gondii chicken isolate (lane 1). Molecular weight standards in kDa (Lane St)

Fig. 2(a-b): Diagnostic potentials of isolated fraction and crude extract of T. gondii chicken tachyzoites local isolate against infected (a) Chicken sera and (b) Human sera

Fig. 3: Diagnostic potentials of affinity isolated fraction of T. gondii chicken local isolate in diagnosis of chicken toxoplasmosis

Fig. 4:Diagnostic activity of affinity isolated fraction of T. gondii chicken local isolate in diagnosis of human toxoplasmosis and other parasitic infections

Table 1: Sensitivity, specificity, PPV, NPV and diagnostic accuracy percentages of isolated fraction against infected human sera by ELISA
ELISA: Enzyme linked immunosorbent assay, PPV: Positive predictive value and NPV: Negative predictive value

Also, it was succeeded in detection of toxoplasmosis in 30 patient sera infected with Toxoplasma. Isolated fraction was cross-reacted with 2 out of 5 patient infected with fascioliasis and 3 out of 10 human schistosomiasis. While, it did not react with human sera infected with Amoeba and hydatid cyst. Fraction recorded 100% sensitivity and 84.4% specificity (Fig. 4 and Table 1). Also, PPV%, NPV% and diagnostic accuracy percentage were 85.7, 100 and 91.9%, respectively (Table 1).

DISCUSSION

Due to significant medical and veterinary problems caused by T. gondii, the present study focused on accurate diagnosis of toxoplasmosis. Accurate diagnosis of the disease is considered a key to resolve the problem. So, utilization of specific antigen and a sensitive method is essential. In previous study in this laboratory, purified fraction of T. gondii horse derived antigen proved high efficacy in diagnosis of human toxoplasmosis (Hassan et al., 2012). Moreover, affinity purified fraction of T. gondii camel isolate was successfully utilized in diagnosis of cattle toxoplasmosis (Toaleb et al., 2013). Recently, chicken T. gondii crude tachyzoites antigen proved success in diagnosis of chicken and human toxoplasmosis (Hassan et al., 2015). This last result in addition to previous ones directed this attention to isolate the components, which responsible for this success. So, CNBr-Sepharose 4B affinity column chromatography was adopted for purification of chicken tachyzoites crude antigen using naturally infected chicken sera.

The purified fraction proved higher diagnostic potency compared with crude antigen in detection of Toxoplasma antibodies in two fold serially diluted chicken and human sera. The fraction resolved into two bands of molecular weight 116 and 83 kDa by SDS-PAGE. This result confirms our earlier observation that two bands of 116 and 83 kDa were detected in crude chicken T. gondii tachyzoites antigen by immunoblot assay using infected chicken serum (Hassan et al., 2015). A single band of molecular weight 116 kDa was previously detected in affinity purified fraction of RH antigen and successfully utilized in diagnosis of mice toxoplasmosis (Hassanain et al., 2013).

Also 116 kDa was detected in RH T. gondii antigen using Toxoplasma infected chicken serum (Hassan et al., 2015). So, 116 kDa is behind the success of chicken T. gondii fraction in the diagnosis of human toxoplasmosis and participate in the diagnosis of chicken toxoplasmosis together with 83 kDa. The role of T. gondii infected chicken in the epidemiology of human toxoplasmosis is previously documented (Aboelhadid et al., 2013). Where, higher seroprevalence of human toxoplasmosis in poultry contacts (37.5%) was recorded than non-poultry contacts (30.5%). This is in addition to the fact that routes to human toxoplasmosis include ingestion of uncooked meat of animals or birds containing tissue cysts (Dubey et al., 2004, 2010). The capability of chicken T. gondii tachyzoites to infect human is partially explained, presently by the existence of common components between both isolates as 116 kDa. In the present study the isolated fraction was used in detection of chicken toxoplasmosis and proved 90.1%. Previously, a total rate of 68.8% was recorded in six Egyptian governorates by ELISA but using crude chicken tachyzoites antigen (Barakat et al., 2012).

Recently, this study recorded chicken toxoplasmosis percentage of 78.12% by ELISA, in which crude Toxoplasma tachyzoites antigen was adopted (Hassan et al., 2015). Differences in prevalence rates are attributed to multiple reasons as sample size, area of study, adopted antigens and assays in the diagnosis. Where, the isolated immunogenic fraction is more potent in diagnosis than crude extract and ELISA is more sensitive than MAT. The high infection percentage in the present study was matched with other results of Dubey (2010a) and Dubey et al. (2010), where they found that toxoplasmosis may be reached to 100% in backyard chicken. Also, in the present study affinity purified fraction succeeded in detecting Toxoplasma antibodies in 30 infected human serum samples with Toxoplasma, recording 100% sensitivity. While its specificity was 84.4%, where it crosses-reacted with Fasciola antibodies. The present results were in harmony with that previous results surveyed in Egypt by Toaleb et al. (2014) and Shaapan et al. (2015) recorded cross reactivity between F. gigantica and T. gondii. Moreover, this cross-reactivity may be due to presence of a band of molecular weight 86 kDa in F. gigantica somatic antigen (Maleewong et al., 1997) and a band of 115 kDa in Fasciola hepatica somatic antigen (Elsagheir et al., 2015).

CONCLUSION

The present study clearly authenticated partially purified chicken tachyzoites antigen proved high diagnostic activity of chicken and human toxoplasmosis with 100% sensitivity and 84.4% specificity.

ACKNOWLEDGMENT

The authors wish to thank the members of Parasitology Department of Kasr Al-Ainy Faculty of Medicine, Cairo University, Egypt, who contributed greatly to this study by collecting the human serum samples.

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