Comparison of electrophoretic pattern of Linguatula serrata proteins obtained from domestic ruminants and dogs by SDS-PAGE

The phylum Pentastomida contains a highly specialized taxon with more than 120 species which infect the respiratory tracts of reptiles and other vertebrates. The species of Pentastomida, a still-enigmatic group of worm-like, linguatula serrata belongs to the bloodsucking parasites that inhabit the upper respiratory tract of terrestrial, carnivorous vertebrates, mostly reptiles and birds; L. serrata, commonly called tongue worms, typically inhabit canids and felids. The intermediate hosts of these parasites are usually sheep, cattle, other ruminants or rodents. The hosts ingest the eggs, and the first instar larva hatches within their intestines, penetrates the mucosa, and retreats into the tissue, where it encysts and molts to the third larval stage. The parasite is tongue shaped, lightly convex dorsally and flattened ventrally. Males measure 1.8–2 cm, while females measure 8–13 cm in length. The nymph of L. serrata is characterised by the possession of a saw-like cuticle with multiple transverse grooves, a ventral buccal apparatus with two pairs of peribuccal hooks and acidophilic glands, a complete digestive system and primordial genital organs. Moreover, in degraded nymphs, the hooks and remnants of the cuticle are often the only findings that allow the diagnosis of the infestation. Humans may be infected with linguatula either by ingestion of nymphs of L. serrate resulting in a condition called nasopharyngeal linguatulosis or Halzoun syndrome or by ingestion of infective eggs which develop in internal organs resulting in visceral linguatulosis. Halzoun syndrome is a rare clinical disease that manifests as an acute allergic-like reaction involving the upper respiratory tract and nasopharyngeal mucosa after the consumption of raw sheep or goat liver, a popular food presentation in Lebanon and other countries of the Eastern Mediterranean region. L. serrata is a common human and animal parasite and affects a wide range of animals, including humans, and causes various diseases in humans. In Iran, this parasite reported from definitive and intermediate hosts in different regions. Also some reports from human Halzoun syndrome it exists in Iran. Diagnosis in final host will be done by clinical signs and separating of the parasite eggs in feces, nasal secretions and animal’s saliva. The diagnosis in intermediate hosts will be done by biopsy. Compression of the electrophoretic pattern of adult and nymph L. serrate proteins obtained from different hosts.

In this study, adult male and female of l. serrate were isolated from the upper respiratory tract of dogs killed by road accident and linguatula nymphs isolated from mesenteric lymph nodes of goats, buffaloes, sheep, cattle and camels. Obtained parasites were separately stored. Collected nymphs of the parasite were washed separately 3 times with 0.85% saline (sterile) and phosphate-buffered saline. The nymph and adult Mouth parts parasites were sonicated on ice with ultrasonicator. After preparing the extract, the protein concentration of parasitic samples was measured by Bradford method and finally, by SDS-PAGE method, the electrophoretic pattern of parasite proteins in different ruminants and dogs was investigated. SDS-PAGE of somatic proteins was performed in different percentage polyacrylamide gels using a discontinuous system as described by Laemmli. The prepared proteins were run on SDS–polyacrylamide gels, composed of 4 % resolving gel and 12 % stacking gel, under reducing conditions using the discontinuous buffer system. For size estimation in SDS-PAGE, a prestained protein marker at a range of 11-180KDa molecular weight (PS10 plus) was used.

Electrophoresis of protein extracts from Mouth parts of different groups of nymphs obtained from different ruminants and adult forms of male and female L. serrata by SDS-PAGE showed a wide range of peptide bands between 8 kDa and 200 kDa. Electrophoresis of Mouth parts of larvae somatic proteins of different groups of parasites (separated from goat, cattle, buffalo, sheep and camel) showed 2 very clear peptide bands with approximate weights of 62 and 53 kDa in SDS-PAGE gel. For proteins prepared from the Mouth parts of cattle and buffaloes, the fine peptide bands did not show much density and were not clear. The status of these bands in the proteins prepared from the oral appendages of camel showed a different pattern from that of other ruminants. It differed from other ruminants in that these bands were clearer than those obtained from cattle and buffaloes and weaker than those from goats and sheep. In male and female adult forms (separated from dog), only the peptide band weighing approximately 62 kDa was present. In the present study, as expected, due to the multicellularity of L. serrate, a wide range of peptide bands in the range of 8 to 200 kDa were found in all samples analyzed by SDS-PAGE. However, there were only two peptide bands in the samples prepared from the oral appendages of the parasitic nodules (about 62 kDa band and about 53 kDa band) and one peptide bond in the proteins prepared from the adult male and female oral appendages (About 62 kDa) were well stained and quite clear, indicating a predominance of peptide concentrations in the mentioned bands.

The prevalence of Linguatulosis is important in ruminants because without any specific clinical symptoms, it leads to reduction of animal productions and hidden economic loss, as well as public health hazards. The results show that the presence of a peptide band with an approximate weight of 62 kDa in all study groups may indicate some of its biological characteristics, especially if we encounter different strains of the parasite is decisive and can be useful in designing efficient serological tests to detect infections in different hosts.