Psoroptes spp. are non-burrowing, obligatory astigmatic ectoparasites that mainly feed on skin cells, lymph, lipid emulsions, red blood cells (RBCs), bacteria, and exudates from the epidermis of their living host (10). As a result, infestations can lead to dermatitis, pruritis, and thick scabs on the skin, causing severe scratching, rubbing, irritation, biting, and hair loss (24). In productive animals, such infestations may result in reduced production and general weakness. In extreme cases, death may occur due to secondary bacterial infections and dehydration (21).

Psoroptes (P.) ovis, which is synonymous with P. cuniculi and P. equi, was established by Zahler et al. (26) in their study ”Species of the genus Psoroptes (Acari: Psoroptidae): A taxonomic consideration”. These mites have been reported worldwide and are known to affect various animal species, including rabbits, cattle, buffaloes, horses, and goats (1,17). In recent years, several studies have characterized Psoroptes mites using both morphological and molecular methods to identify their genetic diversity, transmission patterns, and potential zoonotic risk (9,23). Psoroptes are morphologically relatively larger in size and more oval-shaped than other genera of mites (3). Their long legs are projected beyond the edge of the body (7,20). The long segmented pretarsus containing a funnel-shaped sucker is an important morphological identifying feature of this genus (1,20,22).

In South Korea, psoroptic mange in rabbits has been reported since the 1990s, and the prevalence of Psoroptes mites in domestic rabbits has been reported to range from 28.5% to 53.8% (6,12). Previous studies on Psoroptes spp. in South Korea have primarily focused on the morphological features of these mites; however, this mite species has been inadequately molecularly characterized in this region. The cytochrome c oxidase subunit 1 (cox1) gene has been extensively utilized as a molecular marker for species identification and phylogenetic analysis of mites including P. ovis (11,23). The primary objective of this work is to analyze mites obtained from pet rabbits in South Korea through cox1 gene sequencing. The results of this investigation will enhance our understanding of P. ovis mites in South Korea. In addition, the utilization of cox1 gene sequencing for molecular characterization of P. ovis can be considered a valuable method for accurate species identification.

Sample collection

Between June and September 2022, two approximately one-year-old female pet rabbits were examined at the Veterinary Teaching Hospital of Chungbuk National University. The pet owner described the rabbits’ history, stating that they had been purchased from a pet store in South Korea and that the symptoms were first noticed approximately two to three weeks prior to their hospital consultation. The examination aimed to evaluate for signs of scratching at the ears as well as discharge or waxy debris in the ear canals. Ear scrapings were collected from the ear canals of the rabbits using sterile cotton-tipped swabs and forceps (Fig. 1). These samples were placed in plastic containers for further analysis. The mite specimens extracted were categorized into two groups: one for microscopic examination and another for molecular analysis. Samples designated for molecular analysis were preserved in 70% ethanol and stored at –20°C until genomic DNA extraction.

Figure 1. Case of ear mites. (A) Pet rabbit affected by mites, (B) mite-infested ear lesions with waxy debris in the ear canals of a pet rabbit, and (C) collected scrapings from the mite-infested ear canals of a pet rabbit showing crusts.

Microscopic examination

After smearing the ear crusts directly onto clean glass slides, mites were observed under a light microscope. Adult male and female mites were identified based on their morphological characteristics (17,18) and were randomly selected using insect needles and brushes. The mites were then immersed in Visikol TOX (Visikol, NJ, USA) for 72 hours to make the mite specimens transparent. Following the clearing process, the specimens were permanently mounted onto another clean slide glass using polyvinyl alcohol (PVA). The mounted specimens subsequently underwent morphometric analysis using a compound microscope coupled with a computer and camera for morphometric analysis.

DNA extraction

Genomic DNA was extracted from the mites collected from the two rabbits using a QIAamp DNA mini kit (Qiagen, Hilden, Germany). DNA was extracted to enable genetic identification and phylogenetic analysis. The mites were initially subjected to ethanol evaporation, followed by pulverization with 3.2-mm diameter stainless steel beads. The resulting sample was vortexed thoroughly with 180 μL of ATL buffer. Twenty microliters of proteinase K was subsequently added, and the sample was incubated overnight at 56°C. Afterward, 200 μL of AL buffer (Qiagen) was added, and the mixture was incubated at 70°C for 10 min. Then, 200 μL of 100% ethanol was added, the solution was filtered through a spin column, and the DNA was washed with AW1 and AW2 buffers (Qiagen). Finally, the DNA was eluted with 200 μL of AE buffer (Qiagen) and stored at –20°C until PCR.

Molecular identification

The primers used to amplify the mitochondrial cox1 gene were COF14 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and COR72 (5’-TAA ACT TCA GGG TGA CCA AAA AAT C-3’) (8,23). The PCR reaction volume was set to 40 μL and consisted of 8 μL of Master Mix (ELPIS-BIOTECH, Daejeon, South Korea), 1 μL of each forward and reverse primer, 26 μL of distilled water, and 4 μL of the DNA template. The PCR protocol for amplifying the cox1 gene included an initial denaturation at 95°C for 3 min to activate the enzyme, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 40°C for 1 min, and extension at 72°C for 30 sec. This was followed by a final extension at 72°C for 5 min. The resulting amplicons were analyzed by electrophoresis using a 1.5% agarose gel.

Phylogenetic analysis

Phylogenetic analysis was performed using Geneious Prime version 2024.0.3. Multiple sequence alignment was performed using the MUSCLE alignment technique. Finally, evolutionary distances were calculated using the maximum composite likelihood approach.

Morphological examinations

The findings indicated that adult male mites (Fig. 2) were slightly oval-shaped, with an average body length of ~556 μm (range: 453-611 μm) and an average width of ~405 μm (range: 335-452 μm) (Table 1) . In contrast, the adult female mites (Fig. 3) exhibited an elongated oval shape and were larger than the adult males, with an average body length of ~665 μm (range: 492-834 μm) and an average width of ~437 μm (range: 326-559 μm). Both sexes possessed four pairs of legs; the first three pairs (legs I, II, and III) featured a long segmented pretarsus and ended with ambulacral suckers (Fig. 4). In females, all four pairs of legs were nearly the same size, with leg IV ending in a pair of elongated setae (Fig. 3). However, in males, leg IV was significantly shorter than the other legs and lacked a pretarsus (Fig. 2), marking the primary distinguishing feature between the sexes. Both male and female bodies were observed to have tiny, hair-like structures called setae (propodosomal, metapodosomal and outer opisthosomal setae) on their surface. In males, a gonopore was observed on the metapodosomal region of the ventral surface. On the other hand, females lacked an adanal sucker and opisthosomal lobes in their posterior region. The female anus was located on the ventral side of the opisthosomal area, and the posterior opisthosomal edge had a rounded shape.

Table 1 . Body size of adult male and female Psoroptes mites.

No.	Male			Female		Years	Geographic area	References
	Length (µm)	Width (µm)		Length (µm)	Width (µm)
1	556	405		665	437	2023	South Korea	This Study
2	472	406		568	411	1958	Canada	(19)
3	447	339		587	460	1984	Texas, USA	(25)
4	396	380		536	467	2000	U.K.	(18)

Figure 2. Microscopic image of a male Psoroptes mite isolated from a pet rabbit in South Korea.

Figure 3. Microscopic image of a female Psoroptes mite isolated from a pet rabbit in South Korea.

Figure 4. Microscopic image of the legs of a Psoroptes mite isolated from a pet rabbit in South Korea. The image shows the segmented pretarsus.

Molecular characterization

The DNA sequencing analysis involved the utilization of NCBI BLAST analysis to compare the obtained DNA sequences with existing sequences in the NCBI database. The NCBI BLAST searches showed that our mites’ cox1 gene sequence had a 99% identity with the cox1 sequence associated with accession number MW590279. This identified the mite species as P. ovis, and the sequences have been deposited in GenBank under accession numbers OR985022 and OR985023. This represents the first documentation of mitochondrial cox1 gene sequences for P. ovis isolated from pet rabbits in South Korea. Phylogenetic analysis (Fig. 5) of the cox1 sequences from these South Korean mites indicated they are closely related to global populations of P. ovis. Genetic variations were seen in the phylogenetic tree analysis of the mites from these two rabbits.

Figure 5. Phylogenetic tree of Psoroptes ovis and related mite species based on mitochondrial cox1 gene sequences. The tree highlights the genetic position of P. ovis isolated from pet rabbits in South Korea, with GenBank accession numbers OR985022 and OR985023, as reported for the first time in this study. Phylogenetic analysis was performed using Geneious Prime version 2024.0.3. Multiple sequence alignment was performed using the MUSCLE alignment technique. Bootstrap values (based on 1,000 replicates) are indicated at the branches. The genetic distance model and the method used to construct the tree were Tamura-Nei and Neighbour-Joining, respectively.

This study revealed a detailed methodology for characterizing Psoroptes mites in pet rabbits from South Korea through morphological and molecular analyses. Morphological examinations confirmed the presence of Psoroptes spp., while cox1 gene sequencing shed light on their genetic identity and relationships. Our findings not only highlight the presence of P. ovis in the domestic rabbit population in South Korea but also contribute to the global database on the genetic diversity of this ectoparasite by adding new GenBank accession numbers OR985022 and OR985023.

There has historically been confusion over the naming conventions within the Psoroptes genus. It was initially suggested that P. cuniculi, P. ovis, and P. equi be synonymized under P. equi (26). However, subsequent studies recognized P. ovis as the valid scientific name, treating P. cuniculi, P. equi, and others as synonyms (14,22). Morphological analysis revealed that all Psoroptes mites show the distinctive feature of a terminal sucker on a long, jointed pretarsus, which is a trait specific to the genus (2,18,19,22). Notably, our observations suggest a consistent morphological profile with global P. ovis populations, underscoring the reliability of morphological criteria for initial identification. In addition, male and female mites exhibited differences in size, shape, and reproductive organs, with males typically being smaller than females, corroborating the findings of previous research (4,18). These mites predominantly affect the ears, causing intense scratching in rabbits due to inflammatory responses to the mites’ secretions (5,13), and feed on the blood from the resulting scratches (16). This study (Table 1) observed larger mites than observed in other studies, which may be due to increased blood consumption from pet rabbits (15,25).

Genetic analysis using the cox1 gene confirmed the mite species as P. ovis. Previous research has highlighted the reliability of the cox1 gene as a molecular marker for species identification and phylogenetic analysis in mites (11,23). The high sequence identity (99%) with the previously reported sequence (MW590279) of P. ovis from the NCBI database emphasizes the accuracy of this molecular approach. Moreover, the deposition of the sequences OR985022 and OR985023 in GenBank enriches the global database, contributing to the broader scientific community’s resources for genetic comparison and evolutionary studies of Psoroptes spp.

The phylogenetic analysis showed that the P. ovis mites collected from these pet rabbits in South Korea cluster closely with other P. ovis samples from different hosts and countries, indicating a strong genetic relationship among these organisms. Notably, the P. ovis mites from pet rabbit-1 (OR985022) and pet rabbit-2 (OR985023) in South Korea form a monophyletic group with high bootstrap support, suggesting a high level of genetic similarity between these two samples.

In the context of the tree, the P. ovis species are part of a large clade that includes mites that infest sheep from both the USA and Canada as well as other P. ovis samples from rabbits and sheep in China. This suggests that there might be minimal genetic differentiation among P. ovis mites that infect different host species or that are from different geographical regions, which could be due to recent spread or shared evolutionary pressures across these populations. Other species on this tree include Chorioptes texanus, found in cows from China, and various Proctophyllodes and Mesalgoides species associated with birds from Canada, Spain, and the USA. In addition, Sarcoptes scabiei, another significant mite species that infests rabbits and swine in China, is represented and forms a distinct clade, underscoring its genetic differences from the P. ovis species. The bootstrap values at each node indicate the level of confidence in the topology of the tree based on the genetic data. The scale bar at the bottom left indicates genetic distance. Overall, the tree reveals the genetic distances and relationships between the mites in this study, and the two samples from South Korea add new data to the understanding of the genetic diversity of P. ovis. The impact of P. ovis on pet rabbits is significant, as demonstrated by the clinical signs observed in the infected rabbits from which the mites were collected. Severe scratching, irritation, and potential secondary infections raise welfare concerns and highlight the need for effective treatment and control strategies. Our study supports the use of cox1 gene sequencing as a valuable diagnostic and research tool that can facilitate the development of targeted interventions to mitigate the impact of P. ovis on rabbit populations.

There are several limitations to our study that warrant consideration. The sample size was relatively small, and the study was limited to pet rabbits from a specific region in South Korea. Future research should aim to include a larger and more diverse sample of rabbits that potentially encompasses both domestic and wild populations to better elucidate the prevalence, genetic diversity, and epidemiology of P. ovis infestations.

This study revealed the morphological and molecular characteristics of P. ovis mites found in pet rabbits, as evidenced by cox1 gene sequencing performed for the first time in South Korea, and added new genetic data to the global database. The findings underscore the importance of precise species identification for diagnosing psoroptic mange in pet rabbits, which is crucial for developing effective treatment and control strategies. Further research is necessary to design efficient management strategies, elucidate the pathophysiology of P. ovis infestations, and pinpoint potential risk factors in pet rabbits.

The authors have no conflicting interests.