Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatuslarvae

Abstract

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.Keywords: Rhipicephalus (B.) annulatus, serine protease inhibitors, zymography. ResumoCarrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.Palavras-chave: Rhipicephalus (B.) annulatus, inibidores da serina protease, zimografia.

Seasonal distribution and faunistic of ticks in the Alashtar county (Lorestan Province), Iran

“…After very low level of synthetic pyrethroid insecticide resistance reported from India, its larvae has been resisted to pyrethroid in Iran [ 36 ] that is the situation is worsening and its risk increases. This tick has also been recorded in some regions of Europe such as Portugal, Spain, Italy and Greece; Americas such as Mexico; and in northern Africa [ 37 ]. R. annulatus ticks were reported from Mazandaran province with 35.77%, 49.0% and 51.3% frequency [ 12 , 17 ].…”

Designing of potential vaccine candidates of fused cathepsin L and tropomyosin genes of Rhipicephalus (Boophilus) annulatus tick larva

“…One of the frequent ticks in the world is Rhipicephalus and the species of Rh.annulatus is abundant in north of Iran (16)(17). This tick leads to economic losses due to sucking blood and transmission of pathogens such as Anaplasma and Babesia in cattle industry (18)(19).…”