First report of Thelazia callipaeda infection in Phortica okadai and wildlife in national nature reserves, China

Background: Thelazia callipaeda is a zoonotic parasitic nematode of the family Thelaziidae, with Phortica okadai as the intermediate host and the only conrmed vector in China. China has the largest number of cases of thelaziosis in humans of the world. It is generally believed that domestic animals (dogs and cats) are the most important reservoir hosts of Thelazia callipaeda and directly threaten humans. At present, there is not much research and attention on the role of wildlife in the transmission cycle of thelaziosis in wildlife nature reserves. Methods: During 2016-2019, we selected four wildlife national nature reserve across the country as monitoring points for Phortica okadai and wildlife, and we chose to use y-trap method for monitoring Phortica okadai density. Morphological analysis of the parasites collected from the conjunctival sac of the wildlife was taken as the rst step, and a specic PCR was used for exact conrmation. Results: In 2019, the density of Phortica okadai in Foping National Nature Reserve of China, increased sharply and infected Phortica okadai were newly found in wildlife nature reserves. Wild giant panda, wild boar, leopard cat, and black bear were newly found to be infected by Thelazia callipaeda (one animal of each species). A total of four worms were collected and one worm was collected from each animals. The morphologic characteristics of the four worms led to their identication as Thelazia callipaeda, which was molecularly conrmed by a specic PCR amplication. Conclusions: This is the rst report of Phortica okadai as well as a variety of wildlife including wild giant panda infected by Thelazia callipaeda in wildlife nature reserves in China. This indicates that there has been a transmission cycle of thelaziosis among wildlife in wildlife nature reserves. The increasing number of case reports in wildlife suggests the likely risk of infection of Thelazia callipaeda in villagers around wildlife nature reserves. report of P. okadai as well as a variety of wildlife including wild giant panda infected by Thelazia callipaeda in wildlife nature reserves in China. This study illustrates the importance of wildlife in vector-borne zoonosis. Further studies need to focus on the assessment of the risk of T. callipaeda infection in villagers around the wildlife nature reserves.


Background
Thelazia callipaeda is a zoonotic parasitic nematode of the family Thelaziidae, with Phortica okadai (Diptera, Drosophilidae, Steganinae) as the intermediate host and the only con rmed vector in China [1][2][3][4][5]. The de nitive host span is large and can be infected in wildlife, domestic animals and humans [6]. T. callipaeda is also a kind of parasitic nematodes that parasitize the conjunctival sac and lacrimal duct of mammals. Its reproductive mode is ovoviparous [7]. It takes Phortica sp as their intermediate host and mammals as their de nitive host, establishing a transmission cycle between mammals and ies. Dogs are the most important reservoir hosts. When P. okadai lick the mammal's eye, the infective larvae of T. callipaeda escape from the P. okadai 's proboscis and invade the conjunctival sac of the mammal [4]. The damage caused by parasitic T. callipaeda on the eyes of animals is different in severity. The damage mechanism is the friction of sharp ring folds on the surface of the worm and the mechanical damage of the eye tissue caused by the adsorption of the mouth sac. In addition, adult worm secretions and excreta stimulate the tissues of the eyes. In clinically, some infected dogs show foreign body sensation, which increases secretions, eyelid edema, conjunctival congestion, in ammation or formation of small ulcers, turbidity of aqueous compartment, and increases intraocular pressure [7,8].
In recent years, many countries have reported cases of human infection with T. callipaeda, and in Italy, Germany, Serbia and other European countries, there are also reports of dogs and wild hosts infected with T. callipaeda [9][10][11]. From 1917 to 2019, 643 cases of thelaziosis in humans have been recorded in China [12,13]. China has the largest number of human cases of thelaziosis in the world. It can be seen that the number of infection cases has increased signi cantly since 1970, and although the number of cases has decreased compared with that from 1970 to 1999 in the past 20 years, it still tends to increase again [12,13]. The increase in the number of P. okadai, coupled with the proliferation of domestic animals (dogs and cats), has led to increased vigilance against T. callipaeda infections.
The infection rate of villagers is obviously higher than that of urban people [13]. This is because the villages environment is more suitable for the survival of P. okadai. From 2016 to 2019, we performed ocular examination of domestic animals (dogs) in the villages around wildlife nature reserves and found that the prevalence rate, in 2019, was as high as 84.62% (88 of 104), higher than that in 2016 (38.05% ,43 of 113), 2017 (53.92%,55 of 102 ) and 2018 (56.25%, 63 of 112).
However, most of wildlife nature reserves are surrounded by villages. Moreover, the number of wildlife in the wildlife nature reserves is far more than the number of domestic animals in the surrounding villages. If the wildlife spread the T. callipaeda on a large scale, it will be a threat to the villagers and domestic animals.
In order to understand the prevalence of T. callipaeda in the wildlife nature reserves and surrounding villages, we selected four wildlife national nature reserve across the country as monitoring points for P. okadai and wildlife, during 2016-2019. The four monitoring points are located in the wild giant panda home range, one of the most complex topographical regions in the world with the most obvious vertical zoning of climate, and home to more than 8000 con rmed species of wildlife and plants. There are villages around these reserves, and some villagers raise domestic animals. These areas have overlaps between the activities of wildlife and domestic animals (dogs and cats) [14]. In addition, these reserves were also selected because they are close to densely populated cities.

Methods
Monitoring points for P. okadai and wildlife

Monitoring P. okadai density and morphological identi cation
According to the "Surveillance methods for vector density -Fly" promulgated by the National Health Commission of the People's Republic of China [15], we chose to use y-trap method for monitoring P. okadai density. 200 traps were placed at each monitoring point, of which 100 were distributed in wildlife home range and the other 100 were distributed in surrounding villages. Prepared fruit mash was put in the traps. Monitoring was done fom April to October. The y traps were placed at 9 am in the middle of each month, and were taken back at 9 am the next day. Because there are many kinds of ies in the traps, P. okadai can be identi ed according to the morphological characteristics. They were taken out for further morphological analysis under microscope. The density of P. okadai was calculated and the infection was identi ed by dissection. Density represents the number of P. okadai per cage for 24h [15].

Sample collection
In 2019, four cases of ocular worm infection in wild giant panda (Additional le 1: Figure S1), wild boar, leopard cat and black bear (one animal of each species) were found during ophthalmic examination under injection anesthesia in FNNR. A total of four worms were collected and one worm was collected from each animals.

Parasite collection and treatment
The worms were removed from the conjunctival sac and placed in a sampling tube containing 70% ethanol. Morphological analysis of the parasites collected was taken as the rst step, and a speci c PCR was used for exact con rmation.

Morphological analysis
The parasites collected from the eyes of each animal were identi ed on the basis of morphologic keys under a light microscopy combined with a camera.

Sequence and Phylogenetic analysis
We extracted genomic DNA of each worm from the conjunctival sac of wild giant panda, wild boar, leopard cat, and black bear with the HiPure Tissue & Blood DNA Kit (MAGEN, China). A partial sequence of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1, 689 bp) was ampli ed by PCR. Amplicons were puri ed by using HiPure Gel Pure Micro Kit (MAGEN, China) and sequenced in an ABI3730XL by using the BigDyeTr v3.1 Cycle Seq Kit (Applied Biosystems, USA). Amplicon sequences were determined in both directions (GenBank accession nos. MN719908, MN719912, MN719913 and MN719914) and performed genetic analyses using available sequences of related nematodes from GenBank and the GISAID database (https://www.gisaid.org). The phylogenetic tree ( Fig. 2) was constructed by using MEGA version 6 (https://www.megasoftware.net) by the neighbor-joining method with 1,000 bootstrap replicates [16][17][18][19].
It can be seen from our monitoring data (Table 1-Table 4) that before 2019 (in 2016, 2017 and 2018), there were fewer P. okadai in wildlife home range than in surrounding villages, and no infected P. okadai were found in the wildlife home range. Only in surrounding villages, a large number of infected P. okadai were found. However, in 2019, the density of P. okadai in FNNR increased sharply and infected P. okadai were newly found in wildlife home range of FNNR. July and August were the two months with the highest distribution density of P. okadai, and also the two months with the highest number of P. okadai with larvae of T. callipaeda (Fig. 3a).
Through morphological observation of the collected parasites, it could be seen that the four worms have cylindrical shape with thin ends, milky white and slightly transparent. All of worms were females. The mean body length and width of the female were 13.7mm and 0.36mm respectively. Under the optical microscope, the buccal capsule, pharynx, and esophagus of the anterior segment of the worm body and its serrated, wrinkled surface and the caudal end of the worm were visible (Fig. 3b, Fig. 3c). In the midsection of the female's uterus, there were larvae and oval eggs (Fig. 3d). According to the morphological characteristics of the worm, it could be identi ed as T. callipaeda.
By the 2% agarose gel electrophoresis, the PCR ampli ed products of each sample showed a DNA band of about 689 bp length, which was consistent with the expected size (Fig. 4). There were no nonspeci c bands. According to alignment and phylogenetic analysis, the neighbor-joining method con rmed that the T. callipaeda cox1 sequences in wild giant panda, wild boar and leopard cat (GenBank accession nos. MN719908, MN719912 and MN719913) obtained clustered with those of T. callipaeda in dogs, cats, and humans from China, Japan and Korea. The sequences from black bears (GenBank accession nos. MN719914) were closely related to those from European animals, as they were clustered together. All cox1 sequences of T. callipaeda were clustered into one large branch, while the cox1 sequences of Onchocerca lupi from USA represent an outgroup (Fig. 2).
Through morphological observation and cox1 gene detection, we can con rm that all four ocular worms (one worm was collected from each animals) infected with wild giant panda (Additional le 1: Figure S1), wild boar, leopard cat and black bear (one animal of each species) are T. callipaeda.

Discussion
It is generally believed that domestic animals are the most important reservoir hosts of T. callipaeda and directly threaten humans [21][22][23][24][25][26]. However, in 2019, wild giant panda, wild boar, leopard cat, and black bear have been found to suffer from the thelaziosis.
In 2019, through the investigation of the intermediate host, it was found that there were a large number of infected intermediate hosts in wildlife home range of FNNR. We believe that thelaziosis has already spread into the wildlife nature reserves. The outbreak of infection of P. okadai in wildlife home range of FNNR shows that this transmission cycle has been established.
Summer is the most active season for the intermediate host of T. callipaeda [27], it is also the season for numerous wildlife species to migrate in the wildlife nature reserves [14,[28][29][30]. The newborn larvae of T. callipaeda are directly produced in the conjunctival sac of wildlife. When the intermediate host, P. okadai, licks the wildlife's eye secretions, it sucks the newborn larvae into its body. After ecdysis of the larvae 2 times, the newborn larvae develop into infective larvae and then enter the head and proboscis of the P. okadai. When P. okadai lick the another specimen of host, the infective larvae of T. callipaeda escape from the P. okadai 's proboscis and invade the conjunctival sac of the wildlife, it usually takes 15 to 20 days to develop into an adult (7). The female T. callipaeda can continue to produce larvae till it is about 35 days old, which are then licked by the intermediate host, P. okadai, and the cycle continues. The infected P. okadai threaten domestic animals and villagers.
Once thelaziosis is widely spread among wildlife, with the frequent villager activities around the wildlife home range, it will inevitably threaten the human beings in turn. If the infection is not controlled, the infected wildlife gradually forms a reservoir host bank among wildlife. When the winter is approaching, wildlife migrates to the villages, causing interactions between wildlife and domestic animals [14,[28][29][30]. The interaction creates an opportunity for the transmission of potential T. callipaeda between wildlife and domestic animals by P. okadai. P. okadai of FNNR has the highest density as compared to other studied wildlife biotopes, which is related to its geographical environment and climate [27]. It is located in Foping county, in the northeast of Hanzhong city, Shaanxi province, belonging to the north sub-tropical climate zone, Qinling mountains in the north and Daba mountains in the south are two barriers, with an altitude of 980-2904 meters. The humid air does not spread easily to the north. The climate is mild and humid. The suitable climate provides a good habitat for P. okadai, which is the vector of T. callipaeda. No P. okadai is found in the WNNR monitoring points. The reason is that the altitude of most local mountain peaks is over 4000 meters and the average annual temperature is 8.5±0.5℃, which seems too low for the survival of P. okadai.
In this study, only four T. callipaeda were collected from the eyes of four wildlife, and one worm was collected from each animals. Due to the wide living range of wildlife and irregular activities, the wildlife must be anesthetized for each collection, which brings great di culties to the collection work. Therefore, in order to understand the actual number and species of wildlife infected, we need to continue to collect and screen in the future.
Although no wildlife infection cases have been presently found in the three reserves other than FNNR, the monitoring of the intermediate host needs to be carried out all the time. At the same time, the domestic animals in villages should be given deworming medicine regularly. Such efforts could monitor the epidemic trend of thelaziosis and weaken the threat to the public.

Conclusions
To the best of our knowledge, this is the rst report of P. okadai as well as a variety of wildlife including wild giant panda infected by Thelazia callipaeda in wildlife nature reserves in China. This study illustrates the importance of wildlife in vector-borne zoonosis. Further studies need to focus on the assessment of the risk of T. callipaeda infection in villagers around the wildlife nature reserves.

Declarations
Acknowledgements This work was supported by China Agricultural University Workstation located in the Foping National Nature Reserve. We gratefully acknowledge Xiaolin Wang, Yiwen He and Yidong He for their help with data analysis, and Chang Yu and Qinghui Mu for help with manuscript preparation.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article. Sequences obtained during the current study are available in the GenBank database with accession numbers MN719908, MN719912, MN719913 and MN719914.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.
Authors' contributions YPJ, ZCL, DGL, and JHL conceived the study. YPJ and ZCL organized the sampling plan. ZCL, JQW, YFW, NJH and LBT were responsible for the collection of cases of wildlife thelaziosis used in this study. ZCL obtained the sequence of Thelazia callipaeda and analysed the results. All of members participated in the capture of Phortica okadai in wildlife nature reserve. YPJ and ZCL drafted the manuscript and all authors critically contributed to its nal version. All authors read and approved the nal manuscript.           The wildlife nature reserves in which the monitoring points of Phortica okadai and wildlife were set up, China, 2016-2019. Abbreviations: FNNR, Foping National Nature Reserve; TNNR, Tangjiahe National Nature Reserve; WNNR, Wolong National Nature Reserve; FTZNNR, Fengtongzhai National Nature Reserve Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.