Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens

Abstract

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

Keywords: Cathepsin L; Cysteine protease; Deg-CPR-1; Dermanyssus gallinae; Poultry red mite; Vaccine candidate.

Figure 1

Phylogenetic tree based on the nucleotide sequences of the open reading frames of the cysteine protease genes in poultry red mites (PRMs, Dermanyssus gallinae), arthropods, including other mites and ticks, chickens, and other species such as insects and invertebrates. The tree was constructed using the maximum-likelihood method with MEGA X software [14]. The numbers on the right indicate the clusters. Cluster 1: This cluster was divided into two sub-clusters. The digestive cysteine proteases from various species were classified into cluster 1–1, and cysteine proteases from PRMs (Deg-CPR-1) belonged to this cluster (bold font). Sub-cluster 1–2 included cathepsin L-like proteases and some cysteine proteases that are proposed to play a role in haemoglobin digestion in ticks (underlined). Cluster 2: This cluster comprised the cysteine proteases and cathepsins S, H, K, and L of chickens. Cluster 3: The cysteine proteases from mites, different from those in cluster 1 were included in this cluster, and another cysteine protease from PRMs, Dg-CatL-1, was also included (bold font).

Figure 2

Assessment of the acaricidal potential of the plasma from chickens immunised with the recombinant cysteine protease protein from PRMs (Deg-CPR-1) against poultry red mites (PRMs, Dermanyssus gallinae). (A) The production of antibody in the plasma from chickens immunised with Deg-CPR-1. Four chickens were immunised with Deg-CPR-1 and three chickens were unimmunised. The plasma was isolated from immunised and unimmunised chickens, and the antibody specific to Deg-CPR-1 in the plasma was detected by western blotting. The arrow head indicates the predicted molecular weight of Deg-CPR-1 (63 kDa). (B and C) The mortality rate of PRMs that were fed blood containing the plasma from immunised chickens was assessed every day for 1 week. Four and three plasma samples isolated from the immunised or unimmunised chickens were used in the in vitro feeding assays, and the total number of blood-feeding PRMs monitored was as follows: immunised: n = 155; unimmunised: n = 89 (B). The total number of PRMs fed blood containing the plasma with high antibody titre or low antibody titre was as follows: high antibody titre: n = 74; low antibody titre: n = 81 (C). The number of dead PRMs was recorded and plotted on the graph to generate Kaplan–Meier curves. Statistical analysis was performed using a log-rank test. Results with p < 0.01 were considered statistically significant.

Figure 3

Enzyme activity of the recombinant cysteine protease proteins from poultry red mites (PRMs, Dermanyssus gallinae) (Deg-CPR-1). The enzyme activity of the peptidase domain of Deg-CPR-1 (Deg-CPR-1(PD)-TF) and the whole region of Deg-CPR-1 without signal peptides (Deg-CPR-1(whole)-TF), including the inhibitor domain, was assessed using a SensoLyte Rh110 Cathepsin L Assay Kit (AnaSpec, Inc., Fremont, CA, USA) to measure cathepsin L activity. The concentrations of recombinant proteins used in each assay are indicated along the x-axis. TF alone was used as a negative control. Protease activity was indicated as relative fluorescence units (RFU). Error bars indicate standard deviations. Results with p < 0.01 were considered significant.

Figure 4

Expression analysis of the cysteine protease gene of poultry red mites (PRMs, Dermanyssus gallinae) (Deg-CPR-1). (A) Expression analysis of Deg-CPR-1 in fed and starved PRMs. Some PRMs were fixed after their transfer to the laboratory, designated as “fed PRMs”, and the remaining PRMs were maintained at 25 °C for 7 weeks to digest the blood they had ingested; these were designated as “starved PRMs”. The Deg-CPR-1 mRNA was detected in PRMs from two different farms by RT-PCR. Fed: fed PRMs, Str.: starved PRMs, NC: negative control (distilled water). (B) Expression analysis of Deg-CPR-1 in PRMs fed blood at different life stages. A portion of the starved PRMs were sorted according to their life stages, namely, protonymphs, deutonymphs, and adults, under a stereomicroscope, based on their morphology and body size, and Deg-CPR-1 mRNA was detected by RT-PCR. PC: positive control (cDNA from PRMs of all stages), PN: protonymph, DN: deutonymph, Ad: adult, NC: negative control (distilled water). (C) Expression analysis of Deg-CPR-1 in different tissues. The tissue samples were collected from starved deutonymphs and adults by laser-capture microdissection, and RT – nested PCR was performed to detect the Deg-CPR-1 mRNA in the midgut, salivary gland, and ovary. PC: positive control (cDNA from PRMs of all stages), MG: midgut, SG: salivary gland, Ov: ovary, NC: negative control (distilled water). The actin gene was amplified as an internal control in all expression analyses.