A Preliminary Study on the Antigenic Proteins of Linguatula serrata Nymphal Stage in Sheep with Visceral Linguatulosis

1. Introduction

Linguatula serrata a parasitic arthropod from Pentastomida phylum, the family Linguatulidae, is a zoonotic parasite with an indirect life cycle. Adults live in the nose of dogs (and rarely of men), the body length of males is about 2 cm, whereas female parasites have a length of 2 cm, respectively. Embryonated eggs are released via nasal discharge and/or feces. If intermediate hosts (including cattle, sheep, goats, and buffalo) swallow eggs, the larva hatches and migrates via blood vessels to viscera (including mesenteric and hepatic lymph nodes, liver, lung, etc. ( 1 , 2 ).

Human linguatuliasis has been reported in certain parts of the world, especially the Middle East, Southeast Asia, Africa, and America ( 3 - 8 ). The prevalence of linguatulosis in human populations is unknown since infections can only be diagnosed in the case of rare ocular involvement ( 9 , 10 ) and incidentally discovered at laparotomy or autopsy ( 4 , 7 , 11 ). Linguatulosis in humans has been reported in different provinces of Iran ( 12 - 15 ).

Respiratory ducts, especially nasal cavities, are the habitat of the adult parasite in canine and feline species ( 2 ). The prevalence of linguatulosis in dogs varies considerably throughout the world and is reported as 76.5%, 38%, and 53% in stray dogs in Iran ( 16 ), India ( 3 ), and Turkey ( 17 ), respectively. The infected dogs usually lack specific clinical signs; therefore, linguatulosis in these animals is usually detected at the surgery or autopsy by the isolation of the parasite from some organs, such as the liver, lungs, and lymph nodes ( 18 ). The natural infection of L. serrata in all domestic ruminants and some carnivorous animals has been reported in different geographical parts of Iran ( 16 , 19 - 26 ). The importance of antibody (Ab) or antigen (Ag) detection in serological methods is of utmost importance in the diagnosis of infections.

Moreover, serological assays could also allow the processing of multiple samples in a short time, which would be useful in health management. There is a paucity of information on excretory-secretory (ESP) and somatic (S) antigens of L. serrata. In light of the aforementioned study, the current study aimed to characterize L. serrata antigens, which may be used as a basis for future studies on the specific immunological diagnosis of visceral linguatulosis in sheep and other animals.

2. Materials and Methods

2.1. Sera and L. serrata Nymph Collection

Positive control sera were prepared from the sheep that were selected to slaughter. Blood sampling was performed before slaughtering the animals. The mesenteric lymph nodes of the slaughtered sheep were separated from the body and examined directly or by stereomicroscope for nymphs of L. serrata. The serum of any animal in which the parasite (nymph) was observed in its lymph nodes was considered positive serum. The collected nymphs were washed several times with tap water and then used for somatic extract or excretory-secretory products. Negative sera were prepared from indoor lambs.

2.2. Preparation of S and ES Proteins

The collected nymphs of the parasite were separately washed three times with 0.85% saline (sterile) and phosphate-buffered saline (PBS, pH=7.2, supplemented with 100U/ml of penicillin G. potassium and 100 mg/ml of streptomycin). The viability of the nymphs was checked under a stereomicroscope. For the preparation of crude somatic extract (S), 250 nymphs were triturated by scalpel, homogenized by ultrasonic homogenizer (Bandelin, Berlin, Germany) in 10 mL mixed of PBS and RPMI (with the same proportion), and then centrifuged at 2000 g for 10 min at 4°C (to eliminate cell debris). The extract was filtered through 0.22 mm filters (Biofil Syringe Filter).

The excretory-secretory product (ESP) was obtained from the culture in vitro of the parasite nymphs. A total of 250 nymphs were placed in a 75 cm2 cell culture flask (Greiner Bio-One, Solingen, Germany), containing 10 ml RPMI-1640 (Bahar Afshan, Iran) with 100U/ml of penicillin G potassium and 100 mg/ml of streptomycin. The flask was incubated in darkness for 24 h in a 5% Co2 atmosphere at 37°C. The supernatants were collected, centrifuged at 2000 g for 10 min at 4°C, and filtered through 0.22 mm filters. To increase the protein concentration, filtrates were concentrated up to three times (3X) under nitrogen flow. Protein concentrations of the S and ESP filtrates were determined by the Bradford method and stored at -20°C until use.

2.3. SDS-PAGE and Immunoblotting

The SDS-PAGE of somatic and excretory-secretory proteins was performed in different percentage polyacrylamide gels (4% stacking,12% resolving gel) using a discontinuous system as described by Laemmli ( 27 ). In brief, 60 µl of each sample was mixed with 30 µl of a non-reducing sample buffer [SDS (10%), Tris-HCl (1M; pH 6.8), glycerol (2%)], boiled for 5 min, centrifuged for 1 min, and supernatants were used for loading. For molecular weight estimation, a pre-stained protein marker broad range (25-180 kDa) from BioRad was also subjected to gel electrophoresis, which was carried out using a Mini-Protean III Cell (Bio-Rad) at 100 V constant voltage for 5 h. The gel was stained for protein visualization with Coomassie blue R-250 (0.1% w/v).

For immunoblotting, the proteins of S and ES products were electrophoresed on 12% SDS-polyacrylamide gels. Proteins from SDS gels were electrotransferred onto nitrocellulose membranes (NC) using a Trans-Blot Cell (Bio-Rad) and transfer buffer (Tris 25 mM, glycine 25 mM, pH 9.0) for 3 h at 60 V. Following that, the transferred proteins were blocked with blocking solution (5% skim milk in PBS) at room temperature for 1 h.

The TNC strips were washed (2-5 min) in the washing buffer (PBS-T: PBS, 0.05% Tween20) and incubated for 1-2 h with specific anti-L. serrata sheep sera that were confirmed by enzyme-linked immunosorbent assay  (ELISA) test (n=36). Control tests were carried out with sera from non-infected lambs (n=4). Four dilutions of sera (1:10, 1:20, 1:40, and 1:80) in diluting buffer (PBS-T, 5% skim milk) were examined four times, and the better dilution was selected for continuing the tests. The strips were washed again (2-5 min) in PBS and developed with anti-sheep IgG peroxidase-conjugated (Sigma- Aldrich, USA) in dilution 1:1000 for 1 h at 37ºC and in a mixture of H2O2-Chlornaphtol for 15 min.

3. Results

The protein concentrations of S and ESP were obtained at 90 and 30µg/ml, respectively. The total somatic extracts analyzed by SDS-PAGE (12% gel) revealed more than 18 protein bands with molecular weights of 25 to more than 180 kDa (some predominant bands, including 25, 32, 36, 45, 48, 57, 67, 70, 75, 84, and 100 kDa) (Figure 1).

Figure 1. SDS-PAGE analysis of somatic extracts (S), excretory- secretory products (ES) of L. serrata. M: molecular weight marker

The Coomassie blue-stained SDS-PAGE of ESP of L. serrata nymphs revealed more than 9 polypeptides, from 24 to more than 100 kDa. Polypeptides with molecular weights of 28, 35, 48, 57, and 74 kDa were the most predominant bands (Figure 1).

The analysis of electrophoretic patterns in immunoblotting of somatic extracts (S) and ESP of L. serrata nymphs with the 1:10 dilution of positive serum samples (from naturally infected to L. seratta) and 1:1000 dilution of anti-sheep IgG conjugate, positive reactions (as visible bands) were obtained. The results revealed 12 antigenic polypeptides (bands) in somatic extracts (S) of L. serrata nymphs with apparent molecular weights of 28, 38, 49, 57, 63, 67, 91, and one band higher than 180 kDa (Figure 2). The results demonstrated three main proteins (bands) in the ESP of L. serrata nymphs with apparent molecular weights of 38, 57, as well as one specific polypeptide band (like to S) with a molecular weight of higher than 180 kDa (Figure 2).

Figure 2. Immunoblot analysis of somatic and excretory-secretory antigens of L. serrata against a pool of sera from sheep infected with L. serrata. M: molecular weight marker; S (+): anti- L. serrata somatic antigen; ES (+): anti- L. serrata excretory-secretory antigen; S (-) and ES (-): negative serum

4. Discussion

Linguatulosis in veterinary medicine has attracted assiduous attention in the countries across the globe, including Iran. The eggs of L. serrata, which contain larvae are ingested by different vertebrate intermediate hosts, such as ruminants and men ( 1 , 2 ). There exist a need for a reliable serodiagnostic test to establish the prevalence of pentastomiasis in men, dogs, and livestock. Antibodies usually reflect the contact of the host with the parasite and infective status of an animal. Serological testing of linguatulosis can be the most practical method for monitoring the exposure status of farms to implement control measures.

Scant information is available on the immunological aspect of L. serrata. The results of the present study pointed to the presence of more than 18 protein bands (some of them with molecular weights of 25, 32, 36, 38, 45, 48, 55, 57, 67, 70, 75, 84, 91, 100,110, 135) in somatic extracts of L. serrata nymphs SDS-PAGE profile. The study by Hajipour, Tavassoli ( 28 ) is the only research available on the profile of somatic proteins of L. serrata nymphs. They demonstrated the presence of six similar protein bands (14.4, 32, 36, 48, 75, and 100 kDa) in somatic proteins of the nymphal stage of L. serrata collected from goats and cattle (with the additional band, 120 kDa in cattle). This discrepancy between the protein profiles of L. serrata nymphs in the stated study and those obtained in the current research can be ascribed to differences in methods and type of hosts ( 28 ).

In the present study, attempts were made to determine diagnostic antigens of L. serrata, which can be used for the specific immunological diagnosis of linguatulosis. Infection diagnosis by demonstrating specific antibodies in an infected animal relies on the use of good quality antigen. Furthermore, a detectable and persistent antibody response in all infected animals to an antigen is the ideal choice for the serological diagnosis of infectious disease ( 29 ). Based on the results of the present study, immunoblot analysis of S and ES products using sera of ovine infected with L. serrata showed reactivity with 12 and 3 main proteins, respectively. Among the bands, three common immunodominant antigenic proteins with a molecular weight of 38, 57, and an antigen of more than 180 kDa were recognized in the somatic extracts (S) and ES products of L. serrata nymphs by the sera of sheep with visceral linguatulosis. Alborzi, Ghorbanpoor ( 30 ) evaluated an ELISA test for the serodiagnosis of parasite infection in sheep by using S and ESP of L. serrata. Although in their study, the results of the sensitivity and specificity of the ELISA were almost the same for S and ES, ES antigens were better than S antigens for the detection of infection in sheep. The findings of both studies pointed to the similarity of antigens in the S and ESP of the parasite; moreover, they confirmed the common immunodominant antigenic proteins determined in the current study.

5. Conclusion

The results of the present study demonstrated three common immunodominant antigenic proteins with molecular weights of 38 and 57, as well as an antigen of more than 180 kDa, in the S and ESP of L. serrata nymphs in sheep with visceral linguatulosisthree. These antigens can be considered prime candidates for future serodiagnosis and immunoprotective studies of the parasite.

Authors' Contribution

Study concept and design: A. R. A.

Acquisition of data: S. B.

Analysis and interpretation of data: F. Kh.

Drafting of the manuscript: A. R. A.

Critical revision of the manuscript for important intellectual content: S. B.

Statistical analysis: F. Kh.

Administrative, technical, and material support: A. R. A.

Ethics

All procedures were approved by the ethics committee of the Shahid Chamran University of Ahvaz, Ahvaz, Iran under project number 2020-75487-45.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

This study was financially supported by Shahid Chamran University of Ahvaz, Ahvaz, Iran.

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