Investigation on Prevalence of Sparganum mansoni Infection in Wild Frogs on Hainan Island of China

Background: Sparganosis is a serious food-borne parasitic zoonosis, which is mainly caused by ingesting or open-wound contact of the frog esh infected by Sparganum mansoni, or even by intake of the water contaminated by the parasite. The purpose of the study was to explore the prevalence of sparganum infection in wild frogs distributed throughout the Hainan Island, which is the largest island of the mainland of China and located at the northwest of the South China Sea, and to analyze the risk in local populations to suffer from sparganosis. Methods: From 2018 to 2020, wild frogs were collected from rural ponds, rivers and farmlands in different cities and counties throughout the Hainan Island. After weighing and marking of the frogs, the sparganums were examined and isolated. The sites of the parasite and the number of infections of each frog were recorded and analyzed by statistics analysis. Results: A total of 1556 of wild frogs were examined and isolated. 201 wild frogs were found to be infected by sparganum, and the natural infection rate of sparganum in wild frogs was 12.92% (201/1556). There were 612 sparganums found in the frogs, and the average was 3.04 per frog, while the highest infection rate of wild frogs was in Baoting, up to 32.93% (27/82). The infection rate of sparganum in wild frogs in the central region of Hainan Island is higher than other regions. Most sparganums were located in the hind legs of frog, and the number of the sparganum infection in the frogs was not related to the species and weight of the frogs. Conclusions: On Hainan Island, sparganum infection in wild frogs is relatively common, and constitutes a potential threat to human. Effective measures should be taken to control the incidence of sparganosis.


Background
Sparganum mansoni is the larvae of Spirometra mansoni, which is also the main stage of infection and causes disease to the human body. Sparganums can invade the brain, eyes, abdominal cavity, spinal cord, and subcutaneous tissues of humans causing a serious parasitic zoonosis known as sparganosis.
The source of sparganums causing the infection comes from many vertebrates, but it is mainly parasitic in the body of frogs and snakes. Frog is the second important intermediate host of Spirometra mansoni, and is a very important source of sparganum infection in human. Human infection is resulted mainly from ingesting raw or incompletely cooked esh of frogs infected by sparganums, or placing frog esh as poultice on open wound for treatment of skin ulcers or eye in ammations [1][2][3].
Sparganosis has a global distribution [4][5][6][7], but most cases occur in East and Southeast Asian countries [8][9][10]. So far, over 1,000 cases of sparganosis have been reported in 25 provinces, province-level municipalities and autonomous regions in china [11][12]. In Hainan Island which is the mainland of the Hainan province, human cases of sparganosis have also been reported [11]. In southern China, eating raw frog meat is a traditional diet for many local people. Sparganum infection in frogs have been reported in a number of provinces in China, such as Jiangxi, Anhui, Guangxi, Henan, Guizhou, Zhejiang, Shanxi and Guangdong [13][14][15][16][17][18][19][20][21][22][23][24]. The natural infection rate of sparganum in frogs varies from region to region, which is related to factors such as the living environment, living habits, and the number of hosts investigated.
The warm and humid weather in Hainan Island is very suitable for the survival of frogs, and the local people have used frogs as food since ancient times, which has created the prerequisites for the spread of sparganosis. Therefore, collecting frogs and separating the Sparganosis mansoni in their bodies is of great signi cance for the epidemiological investigation related to sparganosis. The aims of this study were to explore the prevalence of Sparganum mansoni infection in wild frogs in Hainan Island, and analyze the risk of local population to suffer from sparganosis.

Survey sites
The present study was carried out in Hainan Island located at the northwest of South China Sea. This area was geographically separated from Leizhou Peninsula of the continent of Asia by Qiongzhou Strait,

Inspection of Sparganum mansoni infection
The frogs were weighed, numbered and registered, and then anesthetized and sacri ced in the laboratory.
The Sparganum mansoni in the frog muscles were examined under a stereomicroscope with the naked eyes after stripping off the skin. Once identi ed, the sparganums were removed from the muscle and put in a culture dish containing physiological saline. After measurement and morphological observation, most of the white sparganums appear as at ribbons of 1 cm to 8 cm in length, and the maximum length of some individuals reached 12 cm. They are highly active and able to change shape in physiological saline. The parasitic site of each infected frog and the number of sparganum infection were examined to calculate the extent of the sparganum infection.

Statistical analysis
Original data were recorded and sorted using the Microsoft® Excel, and all statistical analyses were conducted using SPSS 13.0. The numbers of the frogs infected by sparganums collected from different cities and counties were compared by the chi-square test (χ2 ). A p-value less than 0.01 was considered statistically signi cant.

Results
A total of 1556 individual wild frogs were examined collected from all 18 survey sites. It was found that 201 wild frogs were parasitized by Sparganum mansoni. The total natural infection rate was 12.92% (201/1556). There were 612 sparganums found in those frogs, and the average infection rate was 3.04 per frog. The maximum number of sparganums identi ed in a single frog was 22. There was a statistically signi cant difference in the infection rate of sparganums in the wild frogs collected from different cities and counties (χ2 = 97.633, P = 0.000), whereas the highest incidence of infection in wild frogs was in Baoting County, up to 32.93% (27/82) ( Table 1 Among the 1556 sampled frogs, the infection rate of Sparganum mansoni in Rana plancyi was 14.56% (90/618). There were 268 sparganums found in those frogs, and the average infection intensity was 2.98 per frog. The incidence of sparganosis in Rana tigrina was 11.78% (105/891), 323 sparganums were found, and the average infection intensity was 3.08 per frog. In addition, there were 47 frogs not classi ed, the infection rate in which was 12.77% (6/47). Twenty one sparganums were found in these frogs, and the average infection intensity was 3.5 per frog. There was no signi cant difference in the infection of sparganum between different frog species. (χ2 = 2.505, P = 0.286) ( Table 3).
The frogs were divided into 4 groups according to their body weights, namely < 30 g group, 30-60 g group, 60-90 g group, and > 90 g group. The infection rates of Sparganum mansoni in these groups were 14 Fig. 3).
Sparganum mansoni can be widely parasitic in various parts of the frog's body, and it is more common in muscle tissue. By analyzing the data of the 201 frogs infected by sparganums, we found that most sparganums were located in the hindleg muscle, up to 447 parasites with an average infection intensity of 3.08 per frog. The infection rate was also the highest in the hindleg muscles, reaching 72.14% (145/201), followed by 20.89% (42/201) on the back, forelegs 13.93% (28/201), abdomen 10.45% (21/201), and body cavity 3.48% (7/201) ( Table 5, Fig. 4 & Fig. 5).

Discussion
Hainan Island locates in the very south of China, a tropical region. The climate is warm throughout the year, with plentiful rainfalls, proper humidity, and abundant crops in the farmland, which is very suitable for the growth and reproduction of a large number of frogs. It also provides optimal natural conditions for the reproduction and spread of sparganums.
The investigation showed that Sparganum mansoni were isolated in 1,556 wild frogs, and a total of 612 parasites were detected. Among them, up to 22 sparganums were detected in a single frog. The natural infection rate of Sparganum mansoni in wild frogs in Hainan Island is 12.92%, and the average infection intensity was 3.04 per frog, indicating that the sparganums are widely distributed in various regions of the island, and the phenomenon of sparganamu infection in wild frogs is common (Table 1) [15], there are still many people who are not aware of sparganum infection in wild frogs, and perform insu cient protection of sparganosis. Especially in recent years, with the changes of eating habits, more and more people start to eat frog meat. Some local people in Hainan Island have the habit of preying on wild frogs. Although there is no con dent evidence showing that locals eat raw frog meat, insu cient heating or incomplete cooking process may still cause sparganum infection.
In this research, we found that wild frogs in different areas are infected by Sparganum mansoni in different situations (Fig. 1)related to factors such as ecological environment, geographical location, climate, local customs and habits. A total of 341 sparganums were colletected from the central region, which had high rate of sparganum infection in wild frogs compared with other regions ( 19.53%) ( Table 2). The infection rate in Baoting and Ding'an counties located in the central region are higher (32.93%) and (27.48%) than other places, suggesting that people of these two regions have a higher risk of sparganosis than in other regions. The central area of Hainan Island is the birthplace of main rivers on the island, with an average annual rainfall of more than 2,200 mm, rich freshwater resources and good water quality. There are many ponds, reservoirs and rice elds suitable for the living and reproduction of frogs. In Baoting county and Wuzhishan city, the original ecological environment is relatively well preserved, and there are more free-range cats and dogs in the area. Free-range cats and dogs have more chances to take wild frogs as foods, and be infected by sparganums. The eggs in their feces can easily enter the water after being washed by rain, thereby forming a natural circulation and reiterative transmission, which provides a better chance for completion of the life cycle of Spirometra mansoni [20]. The infection rate of sparganum in wild frogs in Qionghai city and Dongfang city is zero, and the infection rate in Changjiang county and Lin'gao county is not high (Fig. 1). This may be related to the fact that the collection point is close to the sea. The sea is relatively far from highly crowded living areas of people, so there are less cats and dogs, which are also important sources of infection of sparganum, and actions should be taken to prevent the feces of cats and dogs to enter the water bodies closely related to human's daily life [25]. It has been reported in literatures that no sparganum infection was found in manually farmed frogs, which may be related to the lack of intermediate hosts required for a complete life cycle of Spirometra mansoni in the farms during manual breeding process, such as cats, dogs, and Cyclopes [26][27]. Frogs manually bred is fed with arti cial foods, the frogs have less chance to contact with the outside world and have less chance of preying on cyclopes. In addition, manual breeding environments are generally sterilized and disinfested, which reduces the infection rate of sparganum [28].
The frogs collected in the study are mainly Rana tigrina and Rana plancyi. The Rana tigrina is distributed in various regions and inhabits near water plants that are not far from the land. The body length of Rana tigrina is 60-120 mm, and the largest can weigh up to 200 g. The Gold-stripped frogs are mainly distributed in Baoting county and Wuzhishan city, they inhabits on aquatic plants in the ditch and is smaller in size. The results of the study showed that no signi cant difference in the infection rate of sparganum between different frog species (Table 3), however the tendency of difference in the infection rate was found among frogs of different weights, though it is not statistically signi cant (Table 4). It was shown that the infection of sparganum is random, and they parasitize in frogs of different species and weights. Therefore, the infection rate is not related to them, but to the growth environments of the frogs. However, the infection intensity of sparganum in large frogs is relatively high (Fig. 3), indicating that the esh of the hypertrophied frogs, which is rich in protein, calcium and phosphorus, especially the muscles of the hindlegs, provides abundant nutrients and plentiful space for the growth and development of sparganums. In addition, large frogs have lived for a longer time, and prolonged exposure to the wild environment will increase the probability of recurrent infection, leading to large scale infection. Sparganum mansoni can be widely parasitized in various locations of the frog's body, and it is more common in muscle tissue. There will be edema in the area infected by sparganums, and some may have congestion or bleeding. The current results showed that the frog's hindleg muscles have the most parasites of sparganums, with 447 ( Fig. 5), and the infection rate is also the highest (72.14%) compared to other tissues, and the average infection intensity was 3.08 per frog ( Table 5). The observation that sparganum is mainly parasitized in the leg muscles of frogs is consistent with the ndings made by other domestic scholars [14,15,17,23,24]. It further indicates that sparganums have strong contractility and movtility, and it mostly extends under the skin or curls to live in the cavities in leg muscles, since frogs' leg muscles are more nutritious and provide better condition for the survival of sparganums.
On Hainan Island, wild frogs are infected by Sparganum mansoni commonly, which is a potential threat to local people. In order to prevent the infection of Sparganum mansoni, it is necessary to raise public awareness of sparganum infection in frogs and the associated risk of sparganosis, advocate healthy diet concepts and habits, and abandon capture, sale and purchase of wild frogs, so as to prevent and decrease the incidence of sparganosis [29]. Declarations