Immunodiagnosis of fascioliasis using sandwich enzyme-linked immunosorbent assay for detection of Fasciola gigantica paramyosin antigen.

BACKGROUND: Many immunological techniques have been developed over years using the different Fasciola antigens for diagnosis of parasitic infection and to replace the parasitological techniques, which are time consuming and usually lack sensitivity and reproducibility.
MATERIALS AND METHODS: In this study, Fasciola gigantica paramyosin (Pmy) antigen was early detected in cattle sera using sandwich enzyme-linked immunosorbent assay (ELISA), to evaluate the Pmy antigen performance in diagnosis. This work was conducted on 135 cattle blood samples, which were classified according to parasitological investigation into, healthy control (30), fascioliasis (75), and other parasites (30) groups.
RESULTS: The sensitivity of Sandwich ELISA was 97.33%, and the specificity was 95%, in comparison with parasitological examination, which recorded 66.66% sensitivity and 100% specificity, respectively.
CONCLUSIONS: It was clear that the native F. gigantica Pmy is considered as a powerful antigen in early immunodiagnosis of fascioliasis, using a highly sensitive and specific sandwich ELISA technique.

INTRODUCTION

Fascioliasis and other plant-borne trematodes zoonoses are well-known helminths parasites of herbivorous animals, which are caused by two trematodes Fasciola hepatica and Fasciola gigantica. This disease is found in many areas as Europe, the Americans, Africa and Asia, which results in major economic losses in agriculture communities.[1] Fasioliasis causes considerable economic losses due to high level of mortalities in cattle because of liver condemnation, which inturn lead to reduction in production of meat, milk, and wool, and increases expenditures for anthelmintics.[2]

Human infection with fascioliasis is reported according to world health organization.[3] In Egypt, fascioliasis, existed since Pharaonic ages,[4] can infect also donkeys and camels as well as herbivorous animals, which will finally infect human leading to serious hepatic pathological sequences. About 830,000 people are infected in Egypt.[5] The clinical manifestations of fascioliasis in humans include fever, abdominal pain, persistent diarrhea, vomiting, and eosinophilia. Fascioliasis can be differentially diagnosed from some other diseases as acute hepatitis, schistosomiasis, visceral toxocariasis, biliary tract diseases and hepatic amoebiasis.[67] Treatment of the disease for a human is a world health organization major concern, using Triclabendazole (TCBZ), which is effective at a single dose in Egypt.[4] Despite that chemotherapy with TCBZ is effective in treatment, it does not prevent reinfection. Therefore, animals in endemic countries must be continuously treated with the drug. This approach is prohibitively expensive for developing countries and furthermore, promotes the threat of drug resistance.[8]

Thus, prevention and control of the disease is important to provide a proper treatment before liver damage occurs; this will be achieved by early diagnosis of the disease and development of a proper vaccine for prophylaxis from infection.[9]

The parasitological diagnosis of fascioliasis is based on identification of eggs in stool, duodenal contents or bile, also by recovery of the adult worm during surgical exploration (after treatment) or at autopsy.[10] However, the eggs may be absent or present in very small number at irregular intervals, hence difficult to be found. Besides, eating raw or uncooked liver of infected animals may lead to transient excretion of eggs in stool. The symptoms of fascioliasis may be present for several weeks before eggs are recovered in stool. Thus, serologic tests by detection of Fasciola antibody are alternative methods of confirming Fascioliasis; however, cross-reactions with other helminthic antigens may confuse the interpretation of the results;[10] However, detection of circulating antigens is more specific using anti-Fasciola antibodies due to no cross reactivity with other parasites.[11]

Immunodiagnosis is mainly based on two main categories. The first one is by the detection of the circulating antibobies against the parasite by binding crude or purified, somatic or excretory/secretory (E/S) antigen. The second one is by the detection of the parasite antigen.[12]

Paramyosin (Pmy) (about 100 kDa) predominantly located in muscle and in some cases in the tegument of invertebrates, including some flatworms that are parasites of human and domestic animals. It has a structure as fibrillar, α-helical, and coiled-coil protein.[13] It is widely distributed among invertebrates but absent in vertebrates. Furthermore, it is considered as immunodominant antigen during infection caused by different flat worms such as Schistosoma mansoni, Echinococcus granulosus, Taenia solium and F. gigantica.[14]

This work aims to evaluate an early immunodiagnosis protocol for fascioliasis using the sandwich enzyme-linked immunosorbent assay (ELISA) to Pmy antigen, which is isolated from adult worm homogenate during parasite invasion to help in finding a proper way that inturn will help in prevention of the transmission of the disease.

MATERIALS AND METHODS

Animals

Sixteen Newzealand white male rabbits, weighing approximately 1.5 kg and about one and half month of age, were examined before the experiments (free from Fasciola and other parasitic infection), and maintained at the Schistosome Biological Supply Program, Theodor Bilharz Research Institute, Giza, Egypt. They were kept under standard laboratory care (at 21°C, 45-55% humidity), and supplied with the filtered drinking water ad libitum, 24% protein and 4% fat diet. Animal experiments have been carried out according to the internationally valid guidelines and ethical conditions.

Parasitological examination

Kato-Katz concentration technique[15] and formal-ether sedimentation technique[16] were performed for all cattle's stool samples in order to identify Fasciola eggs or other helminthic ova.

Preparation of antigen

Preparation of F. gigantica whole worm homogenate

Adult clean F. gigantica worms were homogenized in two volumes (vol.) of 20 mM Tris-Hcl buffer (British Drug Houses (BDH) Chemicals, England) containing 5 mM Phenylmethylsulfonyl fluoride as a protease inhibitor (Sigma-Aldrich, Louis, USA) at 20.000 rpm using Initials for the company janke and Kunkel (IKA) T 20 homogenizer (IKA Labortechnik, Staufen, Germany). The homogenate was centrifuged at 30.000 rpm for 30 min. The entire process of homogenization and centrifugation was performed at 4°C. The supernatant fractions were decanted and assayed for protein content and stored at −20°C until used as crude extracts. Protein content of E/S antigen was measured by the Bio Rad protein assay kit.[17]

Purification of F. gigantica Pmy antigen from whole worm homgenate

Purification was carried out in two steps:

Diethylaminoethyl (DEAE)-Sephadex G-25 and G-200 ion exchange chromatography

Sephadex G-25 and G-200 powder (Amersham Bioscience, Uppsala, Sweden) was swelled in about 300 ml of 0.5 M Tris-HCl buffer (pH 7). After swelling of the DEAE-Sephadex, the initial supernatant was removed and washed extensively with 10 mM Tris-HCl buffer (pH 6.5). Then sample was dialyzed versus the eluting buffer. Pmy is washed out of the gel and its protein content was calculated.[18]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Electrophoresis was performed using the Laemmli system,[19] 12% SDS-PAGE (1 mm) under reducing condition according to Bio-Rad Lab. Model 595, Richmond, CA, USA manufacturer, followed by testing of F. gigantica Pmy antigen for reactivity and specificity by indirect ELISA.[20]

Preparation of pAb against Pmy

Before immunization, rabbit's sera were assayed by ELISA for Fasciola antibodies and cross reactivity with other parasites. According to Guobadia and Fagbemi,[21] rabbits were injected intramuscularly, with 1 mg F. gigantica Pmy antigen mixed 1:1 in complete Freund adjuvant. Then, two booster doses were given, at 1 week intervals after the 1ry injection each was 0.5 mg antigen emulsified in equal volume of incomplete Freund adjuvant.[22] 1 week after the last booster dose, the rabbit's sera were obtained and pAb fraction was purified by 50% ammonium sulphate precipitation method.[23] More purification of pAb was performed by 7% caprilic acid method[24] and finally with gel-filtration.[19] The produced Immunoglobulin (Ig) G appeared in a very high degree of purity except for few serum protein contaminants. Partially purified pAb was further adsorbed with the fetal calf serum (FCS) to eliminate any non-specific binding with bovine antigen.

By modified sandwich ELISA,[20] the reactivity of pAb against different concentrations of F. gigantica Pmy antigen was performed. According to Tijssen and Kurstak,[25] standardization of serial dilutions (2.5, 5, 10, 20 and 40 μg/ml) of purified pAb were evaluated by sandwich ELISA, then, the optimum concentration of pAb was conjugated with horse raddish peroxidase (HRP) by using the periodate method.

Samples preparation

Animals’ samples were collected randomly from naturally infected cattle's during several visits to local abattoir. Blood samples were collected during slaughtering. The livers and gall bladders of animals were checked for the adult flukes. Sera were prepared in 0.1 ml/aliquot, heat-inactivated and stored at −20°C until used.

Immunodiagnosis of naturally infected and control sera by sandwich ELISA

Seventy five samples of naturally infected cattle with F. gigantica and 30 healthy uninfected control samples were tested using sandwich ELISA and was compared with other parasites such as 30 samples infected with S. mansoni, hydatid and Hookworm. All these animals’ samples were collected after detection of adult worms in the dissected liver of naturally infected cattle in local abattoir. Sensitivity and specificity of sandwich ELISA were evaluated.

Sandwich ELISA

The reactivity towards Pmy Fasciola antigen of the antibodies rose, in rabbits, against the purified Pmy antigen, and the level of their reactivity with antigen from other parasites (S. mansoni, hydatid and Hookworm), were explored using the sandwich ELISA, based on the original method of Engvall and Perlmann.[20] Wells of microtitre plates were coated with 100 μl/well different concentrations of purified pAb IgG (2.5, 5, 10, 20 and 30 μg/ml) in 0.06 M carbonate buffer, pH 9.6. The plates were washed three times with washing buffer 0.1 M phosphate buffered saline/Tween (PBS/T), pH 7.4. Then, blocked with 200 μl/well 2.5% FCS (Sigma)/0.1 M PBS/T for 2 h and incubated at 37°C. The plates were washed with washing buffer three times. 100 μl of pooled positive and negative sera, was added individually to each well and incubated for 2 h at 37°C. The plates were washed trice with washing buffer. 100 μl/well of peroxidase-conjugated IgG antibodies of dilution 1/50, 100, 250, 500 and 1000 was dispensed and plates were incubated for 1 h at 37°C, and then were washed five times with washing buffer. Color appearance was carried out by the addition of 100 μl/well substrate buffer and the plates were kept in dark at room temperature for 30 min., then, the enzyme reaction was stopped by 50 μl/well of 8 NH2SO4. The absorbance was measured at 492 nm using ELISA reader. Cut-off value was calculated according to Demerdash et al.,[26] as the mean Optical density (OD) reading of negative control + 3 standard deviation of the mean (mean ± 3 SD).

Statistics

According to Campbell,[27] results were evaluated using the one-way analysis of variance (ANOVA). Comparison between two groups was carried out by the Student's t-test. The data were considered significant if P < 0.05, highly significant if P < 0.01 and very highly significant if P < 0.001.

RESULTS

Purification and reactivity F. gigantica Pmy

Figure 1a shows the optical density (OD)280 profile of the adult F. gigantica worm Pmy antigen fractions obtained following purification of whole F. gigantica worm homogenate by DEAE Sephadex G-25 ion exchange chromatography. The eluted Pmy antigen could be represented by fractions (no. 6-14) with a single peak with maximum OD value equal to 2.903 at fraction number 12.

Figure 1a

Profile of chromatography elution fractions of whole Fasciola gigantica worm homogenate on DEAE-Sephadex G-25 ion exchange chromatography

Pmy antigens fractions were further purified by DEAE-Sephadex G-200 ion exchange chromatography. One peak was obtained representing Pmy with OD value 2.420 at fraction number 8 [Figure 1b].

Figure 1b

Profile of chromatography eluting fractions of whole worm homogenate on DEAE-Sephadex G-200 ion exchange chromatography

The eluted protein fraction was analyzed by 12% SDS-PAGE under reducing conditions showing only one band at 97 kDa, which represented Pmy [Figure 1c].

Figure 1c

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of target paramyosin antigens eluted from affinity chromatography columns. Lane 1: Low molecular weight standard. Lane 2: Whole worm homogenate. Lane 3: Target Paramyosin antigen eluted from Sephadex G-25. Lane 4: Purified Paramyosin antigen eluted from Sephadex G-200

The antigenicity of the purified target Pmy antigen was tested by indirect ELISA technique. Serum samples from animals infected with F. gigantica gave strong reactivity against Fasciola Pmy antigen with mean OD reading equal to 1.317 and very weak cross reactions were recorded with sera from animals infected with other parasites e.g., S. mansoni, hydatid and Hookworm.

Production, purification, and assessment of reactivity of rabbit anti-Fasciola Pmy antigen pAb

Production of anti-Fasciola Pmy antigen pAb was done by injecting New Zealand white rabbits with three doses (Priming, 1st booster and 2nd booster). Test blood samples were withdrawn from New Zealand white rabbits before and after the injection.

Purification of anti-Fasciola Pmy antigen pAb carried out in two steps including ammonium sulphate precipitation followed by 7% caprylic acid precipitation methods.

The purity of IgG pAb after each purification step was assayed by 12% SDS-PAGE under reducing conditions. The purified IgG pAb was represented by L- and H-chain bands at 31 kDa and 53 kDa, respectively. The pAb appears free from other proteins [Figure 2]. The reactivity of the purified pAb was tested by indirect ELISA technique after each immunizing dose [Figure 3].

Figure 2

12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of paramyosin of anti-Fasciola gigantica immunoglobulin G polyclonal antibodies (IgG pAb) before and after purification (stained with Comassie blue). Lane 1: Molecular weight of standard protein. Lane 2: Crude anti-Fasciola gigantica paramyosin IgG pAb. Lane 3: Precipitated proteins after 50% ammonium sulfate treatment. Lane 4: Purified IgG pAb after 7% caprylic acid treatment

Figure 3

Reactivity of rabbit anti-Fasciola antibodies against Fasciola paramyosin by indirect enzyme-linked immunosorbent assay

Sandwich ELISA indicated the reactivity of pAb as strong positivity to Fasciola Pmy until 1:250 dilutions. The optimization of various reagents was assayed by sandwich ELISA. The optimum Pmy concentration used was 3 μg/ml; the purified IgG pAb was 10 μg/ml, whereas conjugated IgG pAb was 25 μg/ml.

Stool analysis for Fasciola infection (Kato)

Parasitological examination of cattle stool by Kato-Katz technique for detection and Fasciola eggs count was performed in a total of 135 sheep. Results depicted in Table 1 showed that, Fasciola worms were evident in livers of 75 cattle while Fasciola eggs were detected in only 33 cattle (44%) with egg load ranging from 61 ± 18.30 egg/g to 64 ± 18.30 egg/g stool. Other parasites (S. mansoni, Hydatid and Hookworm) were detected in 30 cattle. Presence of Fasciola worms in liver was used as a gold standard in succeeding experiments.

Table 1

Measurement of intensity of Fasciola infection in relation to ova count

Detection of circulating Pmy antigen in cattle sera by sandwich ELISA

As depicted in Table 2, it was evident that uninfected control sera were 100% negative from Fasciola circulating Pmy antigen. On the other hand, the OD values of Fasciola infected animals group (1.76 ± 0.406) were significantly higher than both of the uninfected control group (0.198 ± 0.098) and other parasite-infected groups (S. mansoni, Hydatid and Hookworm). Seventy three cases were detected as positive samples of F. gigantica infected animals from 75 cases. These two samples were among the light infection subgroup, and the sensitivity of the assay was 97.33% and 95% specificity. On screening the sera of cattle infected by other parasites, it was evident that there was a degree of cross reactivity. Hydatid showed the highest cross reactivity, followed by S. mansoni, whereas, Hookworm show no cross reactivity to the Fasciola infection.

Table 2

Detection of circulating Fasciola Pmy antigen in sera of naturally infected animals

Sensitivity and specificity of sandwich ELISA for detection of Pmy versus stool analysis

Sensitivity percentage of stool analysis was 66.66%, whereas, for detection of serum Pmy antigen by sandwich ELISA demonstrated a higher sensitivity (97.33%). For specificity, stool analysis scored the highest percentage (100%), in comparison to sandwich ELISA, which gave 95% for serum Pmy antigen [Table 3].

Table 3

Sensitivity and specificity percentage of sandwich ELISA used for detection of Fasciola Pmy antigen in serum samples of Fasciola infected animals compared with parasitological examination

DISCUSSION

Fascioliasis, an infectious parasitic disease caused by F. hepatica or F. gigantica affects millions of people world-wide. Up to 17 million people are infected and around 91.1 million are at risk of infection.[28] Today, fascioliasis is recognized as an emerging and re-emerging vector (ungulate)-borne disease with the widest latitudinal, longitudinal, and altitudinal distribution known for any zoonotic disease. Hence, the World Health Organization has classified fascioliasis as an important human parasitic disease that merits international attention.[1] Humans are often infected in communities where there is close human–ruminant interaction, such as in some South American communities, Egypt and Iran.[29]

TCBZ is the only effective drug against early stages of the parasite; however, resistance to it has been extensively reported.[30] Diagnosis in human is based on the findings the characteristic eggs in stool and duodenal fluids;[31] however, negative results do not rule out infection, especially in endemic areas or in field surveys undertaken for exposed agricultural community personnel.[32] Other control measures such as early diagnosis and vaccination should be developed for sustainable control of this disease that will be a cheaper, more efficient and reliable long-term solution for the prevention of infection and eradication of its transmission.

The tegument of bile-dwelling F. gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. Hence, some studies reported that tegument can work as a basis for the development of immunodiagnosis and vaccine production.[33]

This study was done to evaluate a highly sensitive and specific antigen, Pmy, which may be used for early diagnosis of fascioliasis. This aim was achieved through detection of Pmy antigen in naturally infected cattle sera using the sandwich ELISA technique to help in prevention of the infection and to eradicate its transmission.

In the present study, the purification procedure of antigen was carried out by two steps: DEAE-Sephadex G-25 and G-200 ion exchange chromatography.[18] The yield was purified Pmy antigen with protein content equal to 2.6 mg/ml, as a single band at 97 kDa by reducing SDS-PAGE. The results were reasonable with that of Cancela et al.,[13] who purified, characterized and immunolocalized the Pmy from adult stage of F. hepatica.

The preparation of pAb against Fasciola Pmy antigen was carried out by immunization of rabbits with purified Pmy antigen, pAb was purified by; ammonium sulfate precipitation, and caprylic acid treatment and was improved with protein content 10 mg/ml, mainly gamma protein as a double band at 31 kDa and 53 kDa by reducing SDS-PAGE. These yields were valuable as purified Ig following similar purification procedures by Perosa et al.,[34] to purify the human pAb.

The activity of purified pAb was detected by indirect ELISA. It was found that pAb was highly sensitive, specific, and reliable for the detection of circulating Pmy antigen.

The purified anti-Fasciola Pmy pAb was labeled with HRP conjugate (periodate method),[25] and used as a conjugate in the sandwich ELISA to immunodiagnose the fascioliasis infection in cattle. These data were confirmed with El Amir et al.,[35] who labeled pAb against Fasciola E/S antigens to demonstrate the presence of highly reactive epitopes on the different life cycle stages of F. gigantica, as well as on the different organs and peripheral blood mononuclear cells (PBMNCs) of naturally infected cattle.

Recent studies in fascioliasis focused on the importance of ELISA in diagnosis of animal fascioliasis and as it become the most widely used test, because of its simplicity, reliability and easy mechanization.[2] Standardization of the sandwich ELISA showed that, 10 μg/ml/well as the optimum concentration of coating with purified pAb and 25 μg/ml the optimum working concentration of peroxidase conjugated anti-Fasciola Pmy pAb. The lower detection limit of the assay was 2.5 μg/ml Fasciola Pmy antigen which detect high efficacy of ELISA technique in this study. These data were confirmed with El Amir et al.,[36] who evaluated the diagnostic potential of different immunological techniques using F. gigantica E/S antigens in sheep, where the standardization of sandwich ELISA showed that 20 mg/dl/well, as the optimum concentration of coating with purified pAb and 10 mg/dl the optimum working concentration of peroxidase conjugated anti-E/S Fasciola pAb. Although, the lower detection limit of the assay was 0.3 μg/ml Fasciola E/S antigens.

In the present study, 135 blood samples were collected from naturally infected cattle and classified to fascioliasis (n = 75), healthy control (n = 30), and other parasites (n = 30) (10 S. mansoni, 10 Hydatid and 10 Hookworm). According to parasitological investigations (egg count/g), fascioliasis group was classified into heavy infection (n = 33) (64 epg), and light infection (n = 17) (25 epg) although 25 animals gave false negative results.

Rabia et al.,[37] used Kato-Katz concentration and Formal-Ether sedimentation techniques for 152 stool samples of sheep in order to identify Fasciola eggs or other helminthic ova. Fasciola eggs were detected in 78 sheep (80.62%) with egg load ranging from 18 to 80 egg/g stool while other parasites including schistosomiasis, echinococcosis, anclystomiasis, and ascariasis were detected in 30 sheep. Furthermore, El-Aziz et al.,[38] collected 200 blood and fecal samples from 114 Egyptian buffaloes, 68 native breed cattle and 18 imported cattle, they were examined parasitologically for Fasciola infection, the results revealed that 38 buffaloes (33.33%) and 18 native breed cattle (26.50%) were positive although all imported cattle were free from Fasciola.

A Several F. gigantica antigens with immunodiagnostic potential have been identified in preparations of the flukes and their E/S products.[39] The majority of antigenic proteins derived from the surface membrane and the tegument are of 97, 66, 58, 54, 47 and 14 kDa. While, those released from the cecum are 27 kDa and 26 kDa. These antigenic proteins include antioxidant enzyme, glutathione S-transferase (GST), fatty acid binding protein (FABP), membrane protein, hemoprotein, and cystein proteinases (CP), as well as, muscle protein Pmy[40] that can work as a basis for the developments of immunodiagnosis and vaccine.

A monoclonal antibodies-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen in sera of mice infected with F. gigantica, The sandwich ELISA exhibited sensitivity and specificity at 94.55% and 100%, respectively that could be used for early diagnosis of fascioliasis.[41] Furthermore, Hammouda et al.,[42] were assessed the cure of human fascioliasis after TCBZ treatment by detection of Fasciola antigens, using a modified double antibody sandwich ELISA, the results showed that, the antigens were detected in the sera of all studied cases before treatment while no antigens were detected 6 months after treatment. These results suggested that, antigens’ detection provides an accurate tool for diagnosis as well as assessment of cure. The goal of the present study was to develop a diagnostic antigen for early fascioliasis, and this will be achieved by detection of Pmy antigen using the sandwich ELISA.

In this study, the sensitivity and specificity of sandwich ELISA for detection of Pmy antigen in sera was 97.33% and 95%, in comparison to the parasitological examination which gave 66.66% and 100%, respectively. These results indicating highly sensitivity and specificity of sandwich ELISA for immunodiagnosis of fascioliasis. The importance of such fact is that clinically it is vital to diagnose fascioliasis as early as possible in serum to avoid all the complications of such a disease. This is in agreement with EL Ridi et al.,[43] who reported the most prominent nine bands of F. gigantica E/S (62-60, 40, 30, and 28 kDa). The data indicated that E/S-based indirect ELISA reached 100% sensitivity and specificity in immunodiagnosis of sheep fascioliasis. Also, Kumar et al.,[44] evaluated F. gigantica somatic antigen 27 kDa for potential detection of F. gigantica and F. hepatica infection in buffaloes by indirect ELISA, with 81% sensitivity and 97-98% specificity.

There is a degree of cross reactivity that was revealed in the present study between F. gigantica Pmy antigen and other parasites with varying degrees, mainly schistosomiasis and hydatid. This is in agreement with Dalimi et al.,[45] who reported that cross reactive antigens were shared between Fasciola, schistosomiasis, echinococcosis, as well as hydatidosis.

In the present study, the detection of Pmy antigen in naturally-infected cattle sera by sandwich ELISA showed an increase in mean OD readings from light to heavy infection. In other parasite groups, there is a degree of cross-reactivity observed with Hydatid, followed by Schistosoma, while there is no cross-reactivity with Hookworms. Data revealed that, sandwich ELISA is a suitable technique for diagnosis of fascioliasis infections with a lowest degree of cross-reactivity with other parasites by detection of circulating Pmy antigen in the sera.

In conclusion, these studies proved that native heterologous F. gigantica Pmy significantly provide an early diagnosis of fascioliasis in naturally infected cattle. Early diagnosis using the sandwich ELISA is highly sensitive and specific screening immunodiagnostic technique than parasitological examination.