Characterization of a Novel Cysteine Protease Inhibitor from Poultry Red Mites: Potential Vaccine for Chickens

Abstract

Poultry red mite (PRM; Dermanyssus gallinae) is a hazardous, blood-sucking ectoparasite of birds that constitutes a threat to poultry farming worldwide. Acaricides, commonly used in poultry farms to prevent PRMs, are not effective because of the rapid emergence of acaricide-resistant PRMs. However, vaccination may be a promising strategy to control PRM. We identified a novel cystatin-like molecule in PRMs: Dg-Cys. Dg-Cys mRNA expression was detected in the midgut and ovaries, in all stages of life. The PRM nymphs that were artificially fed with the plasma from chickens that were immunized with Dg-Cys in vitro had a significantly reduced reproductive capacity and survival rate. Moreover, combination of Dg-Cys with other antigen candidates, like copper transporter 1 or adipocyte plasma membrane-associated protein, enhanced vaccine efficacies. vaccination and its application as an antigen for cocktail vaccines could be an effective strategy to reduce the damage caused by PRMs in poultry farming.

Analysis of gene expression in poultry red mite, <i>Dermanyssus gallinae</i>, by RNAscope <i>in situ</i> hybridization

“…On the other hand, the probe specific to the Dg-Cys gene successfully detected its expression in the intestinal tract and ovaries ( Fig. 2B ), consistent with a previous report [ 8 ]. These data suggest that the RNAscope ISH assay can be applied for expression analysis in different tissues.…”

“…Cystatin is an inhibitor of cysteine proteases and is involved in the regulation of tick-host interactions and blood digestion in ticks [ 10 , 21 ]. Our previous study confirmed the expression of Dg-Cys in the digestive tract and ovaries by LCM and RT-PCR [ 8 ].…”

“…In addition, expression analysis of each tissue could clarify the functions and biological importance of the targeted molecules. Previous reports have shown the expression patterns of some molecules in different PRM tissues via immunohistochemical (IHC) analysis [ 16 ] and the combination of laser capture microdissection (LCM) and reverse transcription-PCR (RT-PCR) [ 8 , 9 ]. However, specific antibodies against target molecules are required for IHC analysis, and LCM requires specific and expensive equipment, which is rarely available as a general research equipment, and skills in cutting out tissue sections with a laser and collecting them.…”

“…In the present study, we examined the use of RNAscope ISH assay in PRM samples. We detected Dermanyssus gallinae cathepsin D-1 (Dg-CatD-1) and Dermanyssus gallinae cystatin (Dg-Cys) , and their expressions in different tissues have been previously characterized [ 8 , 16 ]. The expression of Dg-CatD-1 , which was predicted as a molecule related to blood digestion, was analyzed by IHC and observed in multiple tissues, except for the ovary [ 16 ].…”

Vaccination Against Poultry Parasites

“…Other proteins were selected because they are homologue to proteins used in tick vaccines (154,155). Antigen cocktails and whole-mite extracts have also been used (156,157).…”