First Report of Thelazia Callipaeda Infection in Amiota Okadai and Wildlife in Wildlife Home Range, China


 Background: Thelazia callipaeda is a nematode of the sucking nematode genus of the sucking family, with Amiota okadai as the intermediate host and vector in China. China has the largest number of cases of thelaziosis in humans in the world. It is generally believed that domestic animals (dogs and cats) are the most important reservoir host 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 home range.Methods: During 2016-2019, we selected four wildlife national nature reserve across the country as monitoring points for Amiota okadai and wildlife. And we chose to use fly-trap method for monitoring Amiota okadai density. Morphological analysis of the parasites collected from the conjunctival sac of the wildlife was taken as the first step, and a specific PCR was used for exact confirmation.Results: In 2019, the density of Amiota okadai in Foping National Nature Reserve, in China, increased sharply and infected Amiota okadai were newly found in wildlife home range. And it was newly found that wild giant pandas, wild boars, leopard cats, black bears were infected with Thelazia callipaeda. A total of 4 nematodes were collected. The morphologic characteristics of the nematode led to its identification as Thelazia callipaeda, which was molecularly confirmed by a specific PCR amplification.Conclusions: This is the first time in China that Amiota okadai has been reported to be infected with Thelazia callipaeda in wildlife home range, while a variety of wildlife, including wild giant pandas, have been infected. This suggests that there has been a transmission cycle of thelaziosis among wildlife in wildlife home range. This has undoubtedly increased the risk of infection of Thelazia callipaeda in villagers around wildlife home range.


Background
From 1917 to 2019, 643 cases of thelaziosis in human have been recorded in China [1,2]. China has the largest number of cases of thelaziosis in human 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 [1,2]. The increase in the number of A. okadai, coupled with the proliferation of domestic animals (dogs and cats), has led to increased vigilance against T. callipaeda infections.
And the infection rate of villagers is obviously higher than that of urban people [2]. This is because the villages environment is more conducive to the survival of A. okadai. From 2016 to 2019, We conducted ocular examination on domestic animals in the villages around wildlife home range 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, the wildlife home range in china have a better climate for A.okadai, and most of wildlife home range are surrounded by villagers. And the number of wildlife in the wildlife home range 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 home range and surrounding villages, we selected four wildlife national nature reserve across the country as monitoring points for A. okadai and wildlife, during 2016-2019. The four monitoring points are located in the giant panda national park, one of the most complex topographical regions in the world with the most obvious vertical zoning of climate, and home to more than 8000 kinds 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 [3]. In addition, these reserves were also selected because they are close to densely populated cities.
Monitoring A. 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 [4], we chose to use y-trap method for monitoring A. 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. The prepared fruit mud was put in the traps. Monitoring was started from the beginning of spring in April to the end of winter in November. The y traps were placed at 9 am in the middle of each month, and was taken back at 9 am the next day. Because there are many kinds of ies in the traps, A. okadai can be identi ed according to the morphological characteristics and crawling speed of various ies. Then it was taken out for further morphological analysis under microscope. Then the density of A. okadai was calculated and the infection was identi ed by dissection. Density is determined by dividing the number of A.okadai by the number of y traps and represents the number of A. okadai per cage for 24h [4].

Sample collection
In 2019, 4 cases of ocular worm infection in wild giant panda (Fig. 2), wild boar, leopard cat and black bear were found during ophthalmic examination with injection anesthesia in FNNR.

Parasite collection and treatment
The worm was 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 wildlife 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 from the T. callipaeda in the conjunctival sac of wild giant panda, wild boar, leopard cat, black bear with the HiPure Tissue & Blood DNA Kit (MAGEN, China). And 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). 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 was constructed by using MEGA version 6 (https://www.megasoftware.net) by the neighbor-joining method with 1,000 bootstrap replicates.

Results
After morphological observation under microscope, A. okadai body length was measured to be 2.7 ~ 3.6 mm. The compound eye was surrounded by a white band, and there were many brown spots of different sizes and irregular shapes on the back of the chest. There were three black bands at the base, middle, and end of the foot, one of the characteristic signs [5,6]. The dorsal side of the 3rd-5th segment of the abdomen each had a dark brown shape similar to "hill" shaped transverse belt (Fig. 3).
It can be seen from our monitoring data (Table 1-Table 4) that before 2019 (in 2016, 2017 and 2018), there were fewer A. okadai in wildlife home range than in surrounding villages, but no infected A. okadai were found in the wildlife home range. Only in surrounding villages, a large number of infected A. okadai were found. However, in 2019, the density of A. okadai in FNNR increased sharply and infected A. okadai were newly found in wildlife home range. July and August were the two months with the highest distribution density of A. okadai, and the two months with the highest number of A. okadai with the early stage of pre-infection larval of T. callipaeda (Fig. 4a).           Through morphological observation of the collected parasites, it could be seen that under stereoscope, the worm body was a cylindrical shape with thin center at both ends, milky white and slightly transparent. All of worms were female. The mean body length and width of the female were 13.7 mm and 0.36 mm 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. 4b, Fig. 4c). In the midsection of the female's uterus, there were larvae and oval eggs (Fig. 4d), with the mode of reproduction of oviparous. According to the morphological characteristics of the worm, it could be identi ed as T. callipaeda.
According to alignment and phylogenetic analysis, the neighbor-joining method con rmed that the T. callipaeda cox1 sequences in wild giant panda, wild boar, leopard cat (GenBank accession nos. MN719908, MN719912 and MN719913) obtained clustered with that 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 specimens and 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 was clustered into one branch (Fig. 5).

Discussion
It is generally believed that domestic animals are the most important reservoir host of T. callipaeda and directly threaten humans [11][12][13][14][15][16]. However, in 2019, many species of wildlife that have not been reported before have been found to suffer from the thelaziosis. And through the investigation of the intermediate host, in 2019, it was found that there were a large number of infected intermediate hosts in wildlife home range.
We believe that thelaziosis has already spread into the wildlife home range. The emerge of infected A. 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 [17], and it is also the season for many wildlife to migrate to the wildlife home range [3,18,19,20]. The infected wildlife will establish the transmission cycle of thelaziosis between wildlife and A. okadai in wildlife home range. There are nearly 2000 kinds of wildlife in China, and there are about 265 kinds of wildlife in FNNR alone. 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. And, the infected wildlife gradually forms a reservoir host bank among wildlife. When the winter is approaching, wildlife migrates to the villages, and enter the overlapping areas between wildlife and domestic animals [3,18,19,20]. The interaction creates an opportunity for the transmission of potential T. callipaeda between wildlife and domestic animals by A. okadai.
A. okadai of FNNR has the highest density, which is related to its geographical environment and climate [17]. It is located in Foping county, located in the northeast of Hanzhong city, Shaanxi province, belonging to the north sub-tropical climate zone, Qinling mountain in the north and Daba mountain in the south are two barriers, with an altitude of 980-2904 meters. The humid air is not easy to travel to the north. The climate is mild and humid. The suitable climate environment provided a good habitat for the A. okadai, which is the media of T. callipaeda. No intermediate host A. 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 is not conducive to the survival of A. okadai.
Although no wildlife infection cases have been found in the other three reserves (TNNR, WNNR, FTZNNR) and the infected A. okadai was only found in wildlife home range of FNNR and surrounding villages, 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 time in China that A. okadai has been reported to be infected with T. callipaeda in wildlife home range, while a variety of wildlife, including wild giant pandas, have been infected. 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 home range.
Declarations Figure 2 Thelazia callipaeda in the eyes of wild giant panda