Onchocerca Flexuosa. sp. (Nematoda: Filarioidea) in Japanese Wild Sika Deer (Cervus Nippon): Pathological and Molecular Identication of Intradermal Worms

More than thirty species of the genus Onchocerca (Nematoda; Filarioidea) were responsible for onchocercosis (a vector-borne parasitic disease) of wild and domestic ungulates. “Between 2016 and 2017”, the presence of Onchocerca exuosa (Wedl, 1856) was detected and investigated in 17 Japanese Sika deer (Cervus nippon) captured in Gifu and Shiga Prefectures, Japan. The worms were found within characteristics subcutaneous nodules dispersed in different regions of the body of all 17 deer. The all collected nodules were examined stereo-microscopically. The parasites were extracted from the nodules and identied through morphological and histopathological examinations. Molecular identication through sequencing of the following genes; internal transcribed spacer subunit 2 (ITS2)–28S ribosomal RNA (28S rRNA), cytochrome c oxidase subunit 1 (cox1) and mitochondrially encoded NADH dehydrogenase subunit 2 (NAD2) were performed. The histopathological, molecular and phylogenetic analysis demonstrated that, the larial nematode isolated from Gifu and Shiga Prefectures in Japan is O. exuosa. This is the rst report about presence of O. exuosa in Japanese Sika deer (Cervus nippon) in Gifu and Shiga Prefectures.


Introduction
Various species of animals either domesticated (cattle, camels, and horses) or wildlife (deer, serows, and boar), can be infected by various species of Onchocerca all over the world. The parasites result in characteristic lesions in joints with muscular in ammation and lameness in ungulates [1].
Onchocerciasis is an established disease caused by larial worms, which are transmitted by arthropods. Onchocerciasis, caused by O. volvulus, has been reported in Africa and Central/South America [2] resulted in a serious diseases as river blindness, which reported in different parts of the world [3].
Human infection was described in Europe caused by O. jakutensis which is a red deer origin [4]. Several clinical human cases, have been recorded worldwide which transmitted to human from animals: Among these cases, eleven were in Japan [5,6]. Onchocerca exuosa (Wedl, 1856) is a larioid nematode (Spirurida: Onchocercidae), whose primary host is the European red deer (Cervus elaphus) [7]. The adult worms live inside subcutaneous connective tissue free or within rm brous connective tissue nodules.
The nodules are heavily in ltrated by different types of in ammatory cells surrounding the coiled worms.
The micro lariae are located near or far from the adult worms [8].
Routine diagnosis is mostly based upon microscopic examination to determine the morphology of the adult worm and its micro laria. However, Giemsa staining cannot differentiate between the most related species [9]. De nitive diagnosis and control of such parasites are of great health concern, and speci c molecular data will help scientists discriminate between closely related species and will also provide new drug target sites unique to the Onchocerca species.
A major strength of molecular identi cation depending up on speci c DNA barcode, as it is allows correlation of any small part of parasites/organism to a single molecular entity (Molecular Operational Taxonomic Unit, MOTU) [10], and taxonomy skilled personnel does not necessary required in the molecular data analysis. cox1 gene sequences and several others mitochondrial markers genes are widely used for DNA barcoding of metazoans [11,12].
In the present study, we describe the morphological, pathological and molecular characteristics of worms of a novel Onchocerca species present inside subcutaneous nodules in the skin of a Wild Sika Deer (Cervus Nippon) in Gifu and Shiga Prefectures in Japan. The molecular diagnosis based on molecular identi cation of 3 genes; internal transcribed spacer subunit 2 (ITS2)-28S ribosomal RNA (28S rRNA), cytochrome C oxidase subunit 1 (cox1), and NADH dehydrogenase subunit 2 (NAD2). University, Japan. Five subcutaneous nodules from each deer were properly collected, cooled by ice wrapping and the collected nodules were all examined using a stereomicroscope. Parasites were detected in all the examined subcutaneous connective tissues nodules. Worms were removed with a pair of small forcipes, and saline was used for ease in detaching. The collected worms was then morphologically examined, Onchocerca nematodes were suspected to be the infective parasite based on the morphological features [13,14]. The worms collected from each nodule, rinsed in normal saline, and stored in 70% ethyl alcohol and used for parasitological and molecular identi cation. About 50% of the worms were used for parasitological identi cation and the rest of the worms were maintained at − 20°C until used for DNA extraction and PCR analysis.

Material And Methods
Animals used in this study were also reviewed and handled according to code of ethics of Research and Animal Resources Committee, Faculty of Applied biological science, Gifu University, Japan, approved by Gifu university animal experiment committee.

Histopathological examination
Tissue specimens of subcutaneous nodules collected from the infected deer were xed in 10% neutral buffered formalin for histopathological examination. Fixed specimens were routinely processed through dehydration in ascending grades of ethanol, cleaned in xylene, and embedded in para n. Para n sections were obtained and stained with hematoxylin and eosin (H&E) for histopathological examinations.

DNA extraction
Total DNA extractions from worm nodules were performed using the AllPrep® DNA/RNA Mini kit and DNeasy Blood & Tissue Kit (Qiagen, Hilden Germany) according to the manufacturer's instructions. PCR ampli cation: Each DNA extract was used as a template for PCR ampli cation using the speci c larial primers CO1F3-CO1R3 and ND2F1-ND2R1 [15]. In addition, parts of the nuclear ribosomal gene repeat were also ampli ed. The ITS2 region (with short portions of the anking 5.8S and 28S genes) was ampli ed using previously published primers [16]. The PCR technique was performed in a 50-µl volume containing 4 µl of DNA, 4 µl of dNTP, 1 µl (10 µMol) of each forward and reverse primers, 5 µl 10× Ex Taq buffer, 0.25 µl Ex Taq polymerase (Takara, Kyoto, Japan), and 34.75 µl distilled water. The PCR cycle for NAD2 and cox1 genes was as follows: denaturation at 94°C for 3 min; 30-35 cycles of 30 sec at 94°C; 30 s at 50°C, and 1min at 72°C; followed by a nal extension period of 5 min at 72°C. PCR cycle for the ITS2 region (with short portions of the anking 5.8S and 28S genes) was as follows: denaturation at 94°C for 5 min; 35-40 cycles of 94°C for 45 sec, 58°C for 45 sec, and 72°C for 90 sec; followed by a nal extension period at 72°C for 7 min.
All PCRs were performed using the Takara Thermal cycler, Japan. PCR products were visualized and photographed in a 0.75-1% agarose gel containing 0.5 mg/ml ethidium bromide on a Gel Documentation System. DNA Sequencing, sequence alignment, and phylogenic analysis PCR amplicons were puri ed and prepared for sequencing using the QIA quick gel extraction kits® according to the manufacturer's instructions. The puri ed PCR products of all O. exuosa positive samples were sequenced using the AB3500XL Genetic analyzer (Applied Biosystems, HITACHI, Japan) at the genome center of Gifu University, Japan using gene-speci c primers.
The obtained nucleotide sequences of ITS2-28S rRNA, cox1, NAD2 and the deduced amino acid sequences of O. exuosa were edited using the Sequence Scanner Software 2 program (http://www.appliedbiosystems.com). Primer sequences were omitted prior to phylogenetic analysis; the edited sequences were computationally compared with those of other larial reference sequences for determining the homology and were subjected to phylogenetic analyses using the MEGA 7software (www.megasoftware.net/). The phylogenetic trees were generated using the neighbor-joining (N-J) tree method, and the reliability of internal branches was assessed by 1000 bootstrap replications [17,18]. The reference sequences of larial genes were retrieved from the GenBank database, and their accession numbers are listed in (Table. 2).

Results
Gross, morphological and histopathological results: Round, at, ovoid or irregular nodules containing Onchocerca were found in subcutaneous tissues of the back, abdomen and anks of examined deer either in isolated or fused form, giving the appearance of lobulated nodules (cauli ower growth). They were solid and hard, and often surrounded by thick brous connective tissue. Microscope-aided gross and parasitological examination of all collected subcutaneous nodules revealed presence of very thin, long, nematodes identi ed as larial worms. O. exuosa (Wedl 1956) (Order: Spirurida; Family: Onchocercidae, subfamily: Onchocercinae; Genus: Onchocerca). The living worms were transparent and opalescent, yellowish-white larioids. The Esophagus is divided into anterior muscular part and slightly longer posterior glandular part. The length of the females was much longer (about 80 mm) than the males (about 60 mm). The female worms had a rounded anterior ends and its widest diameter was approximately 170-280 µm. The female cuticle was approximately 9 µm thick with external delicate transverse cuticular ridges with small protuberances (Fig. 1A). The males had a distinctive coiled posterior end with caudal alae and reduced number of post-cloacal caudal papillae with two unequal spicules (the left one is longer). The right spicule structure has a prominently dorsal hook. The transverse ridges were 4 µm apart at mid-body and tapering toward both ends.
Histopathological features of H&E stained sections are illustrated in (Fig. 1B, C, & D). The subcutaneous nodules exhibited multiple compartments or cavities containing numerous numbers of nematode sectioned at various orientations surrounded by brosing granulomatous tissue. Most of the nodules were inhabited by several females, with or without micro lariae, and fewer male worms lying in small cavities (Fig. 1B). The nematode body was formed of an outer layer of eosinophilic cuticle and a thick muscle layer. In the pseudocoelom of some worms, two engorged uteri and small-sized intestine were observed. Many developing micro lariae were observed in the uterine cavity (Fig. 1C). Eosinophil, lymphocyte, and plasma cell in ltration was observed in the brous nodules around the worms exhibiting strong broplasias leading to the formation of a parasitic granuloma (Fig. 1C, D). Amorphous eosinophilic material (Splendore-Hoeppli reaction) was observed around some female worms (Fig. 1B). Degenerated worms surrounded by granulomatous reaction consisting of remnants of worms, cell debris, eosinophils, macrophages, lymphocytes, and giant cells with a thick peripheral brous layer were frequently observed (Fig. 1D).

Detection of O. exuosa by PCR
All collected subcutaneous nodules representing the 17 deer under investigation tested positive for cox1, ITS2-28S rRNA, and NAD2 genes segments by conventional PCR. The PCR amplicons from Onchocerca worms yielded around 600 bp for ITS2-28S rRNA, and 1000 bp for both cox1 and NAD2 ( Supplementary   Fig. 1). The PCR products were further sequenced for alignment with the reference larial strains available on gene bank data base, and homology analyses as well as generation of phylogenetic tree were performed.
Homology analysis of nucleotide and deduced amino acid sequences Homology analysis of the nucleotide sequences of the three larial nematode genes cox1, ITS2-28S rRNA, and NAD2 as well as the deduced amino acid sequences of cox1, and NAD2 genes segments showed variable identity with the genes of different Onchocerca species available on the gene bank (Tables 2, 3 Fig. 4).
We compared the nucleotide sequences of Cox1, ITS2-28S rRNA and NAD2 genes sequences of Cox1 and ITS2-28S rRNA genes of our samples from captured deer with those of all Onchocerca species available in GenBank. Cox1 sequence of this study showed high identity (94%) with O. exuosa isolated from Miyazaki Prefecture in Japan and from USA red deer isolates. It also showed 90-91% identity with human O. volvulus isolate obtained from Brazil, cattle O. ochengi isolate obtained from Cameroon, O. ochengi obtained from Miyazaki Prefecture Japan, The novel sequences obtained in the present study also exhibited reduced identity (89.37% − 89.68%, 91.78%) with Onchocerca dewittei japonica and Onchocerca skrjabini respectively using ITS2_28S sequence, and (87-89%) with human larial worms (Wuchereria bancrofti, Brugia timori, B. malayi, and B. pahangi) detected by using NAD2 sequence.
As shown in Table (3), p-distances in nucleotide sequences of cox1 were 0-0.044 for intra-species and 0.038-0.051 for inter-species of Onchocerca. Similarly the results of p-distances in nucleotide sequences of ITS2-28S rRNA and NAD2 genes were showed in tables (3,4) and (supplementary tables 1.2 and 3).

Phylogenetic analyses
Phylogenetic trees for Onchocerca spp were constructed using the NJ and ML methods for the nucleotide sequences of cox1, NAD2, and ITS2-28S rRNA genes (Figs. 2, 3, and 4).

ITS2-28S rRNA region
In the same scene, the phylogenetic tree generated based upon the ITS2-28S rRNA region as shown in (Fig. 2), revealed grouping of the onchocerca spp into two clades, clade 1 divided into two subclades. The

Cox1 gene
The phylogenetic tree generated based upon cox1 gene sequences as shown in (Fig. 3), showed that, the larial species were distributed in two main clades.  (Fig. 4).
The results clearly identi ed that Onchocerca species is the cause of parasitic nodules in the deer under investigation in the present study. As shown in (Table 2,

Discussion
Filariasis, and anthroponotic onchocerciasis have been reported worldwide and have caused problems either in animals or humans [5,19], as many cases of zoonotic onchocercosis are reported [20]. Anthroponotic onchocerciasis has also been documented in Africa and Central/South America and has resulted in visual impediments in the affected individuals [6,21].
In the current study, parasitic nodules in the form of yellowish subcutaneous nodules in various parts of the body have been observed in Japanese Sika deer (Cervus nippon) captured from Gifu and Shiga Prefectures in Japan. These ndings are in agreement with the previously reported onchocerciasis in red deer (Cervus elaphus) in Switzerland [20]. The morphology of the present larioids have the main characteristics of the genus Onchocerca that given by Bain [22] i.e. females have a thickened cuticle with transverse annular ridges on their surface and weakly developed muscular layer.
Histopathological and the measurements analyzed, matched with those given in the description of O. exuosa, carried out by earlier investigators [7,8,23]. The detected long female of O. exuosa is identical to the female of O. jakutensis discovered by Bosch et al [20] from the Swiss red deer. Chronic parasitism in the form of multiple granulations around the worm and marked leukocytic cell in ltration of eosinophils, lymphocytes, macrophages, and plasma cells were observed and these granulomata were found mainly around female worms. Presences of many degenerated worms within several cavities in the nodules are an indication of a strong immunological response against O. exuosa [8,24]. Female worms were found more frequently in the nodules than males, they were embedded in the nodular brous tissue, and most of them were usually immobile due to an early degenerative alteration of their muscles. In contrast, mobility activity of males is higher than females as they can migrate subcutaneously leaving  [27,28], indicated that the phylogeny of the family Onchocercidae based on the previously mentioned genes sequences plays a vital role in the classi cation of larial worms along with the classi cation based upon morphological characters.
In a previous studies, DNA analysis and barcoding based on cox1 and other mitochondrial genes was considered a reliable method for species identi cation of larioid nematodes, with a high coherence with classical taxonomy [29]. In recent studies different approaches to species recognition generated similar results [30], which suggested that integration of traditional and molecular approaches to species recognition is possible [31]. More recently Identi cation of Onchocerca spp using cox1 as a molecular marker is considered accurate as previously indicated for Onchocerca spp [32], as well as other larial species [29]. DNA analysis was considered a good and reliable approach for taxonomical identi cation of larioid nematodes, with a high coherence with classical taxonomy [29].
In the present study, the larial parasite was identi ed as O. exuosa based on traditional and molecular analysis. Phylogenetic analysis based on cox1, ITS2-28S rRNA, and NAD2 gene sequences revealed that only three main groupings were recorded in the family Onchocercidae, viz., Onchocerca spp, Diro laria spp, and Brugia + Wuchereria. Similar conclusion has been reached by [16,33] based upon molecular characteristics and phylogeny of the Filarioidea superfamily. Thus, the phylogenetic aspects of larial worms may be resolved by analysis based on cox1, ITS2-28S rRNA, and NAD2 genes.
In our opinion, the light of the present work suggests that cox1, ITS2-28S rRNA, and NAD2 appeared an appropriate molecular marker for identi cation of larioid nematodes up to species level. Similar suggestion has been reached by using cox1 and 12S rDNA genes for identi cation of larial nematodes up to species level [29]. The phylogenetic analyses of cox1, the ITS2-28S rRNA, and NAD2 genes in that study indicated that O. exuosa isolated from Japanese Sika deer was identical to Onchocerca spp in terms of nucleotide sequences. According to [29], no overlap of intra-and interspeci c nucleotide divergence at the cox1 gene occurs at distance values greater than threshold values (approximately 4.8%). Based upon intra-and interspeci c nucleotide divergences, in this study we calculated the genetic distance between cox1 from two different species, and it was greater than the threshold value of 4.8%. Therefore, our ndings clearly document the conclusion that the present isolate is O. exuosa, which is distinguished from O. dewittei japonica, O. takaokai and other congeneric species at the species level, and has a close a nity to other O. exuosa previously reported.
The sequencing analysis is of great importance to identify the origin of the genus Onchocerca and its relation with other members of the family and this characteristic could be used to medicinally control the infection by targeting a speci c gene. The emergence of more infectious diseases is a signi cant problem for the world economy and public health. Majority of these diseases originate in wildlife and spread to other species including animals and humans, and their incidence has increased signi cantly over the past few decades [34]. Thus, disease surveillance efforts and accurate diagnosis should be applied frequently especially in wildlife to control spread of these pathogens to human or other species.         Phylogeny of lariae and related nematodes based on ITS2 taxa. The GenBank accession numbers for each sequence are shown adjacent to each strain.