Comparative Analysis of Small Mammal and Parasitic Flea Composition in Yulong and Jianchuan Plague Foci in Yunnan Province, China: A Cross-Sectional Study

Plague is caused by Yersinia pestis which a gram-negative
bacterium, usually transmitted by fleas bitten and small mammals
as the main reservoir...


Introduction
Plague is caused by Yersinia pestis which a gram-negative bacterium, usually transmitted by fleas bitten and small mammals as the main reservoir. Plague is one of the most serious infectious diseases in human history. To date, three plague pandemics-the Justinian Pandemic, which started in AD 541, the Black Death, which started in AD 1347, and the Third Modern Pandemic, which started in AD 1772-have caused hundreds of millions of deaths worldwide [1,2]. Plague as a natural epidemic disease, is independent of human and exists in natural foci [3]. The disease continues to be a threat to human health, with plausible outbreaks in Madagascar, sporadically spreading in Tanzania, Vietnam, China, and the United States et al in last decades [4]. According to the WHO(World Health Organization), 2417 cases of plague, including 1854 cases of pneumonic plague and 355 cases of bubonic plague, and 209 deaths were reported in Madagascar between August 1 to November 26, 2017 [5]. This outbreak highlights the notion that plague are exists and continues to pose a threat to human health.
China is one of the most complex plague areas in the world, consisting of twelve types of plague natural foci. In the 21st century, the plague natural foci area in northwest China (such as Inner Mongolia, Gansu) are still active, while other foci area (like Yunnan, Gunangxi, Guizhou, etc.) are relatively stable, only animal plague and sporadic human cases reported [6]. The most serious plague outbreak in China was occurred in the Yunnan province. Currently, the plague epidemic affects approximately 10 million people in Yunnan. Plague is therefore an important public health problem in this province [7]. There are wild rodent plague foci and commensal rodent plague foci existed in Yunnan. The former includes those in Yulong and Jianchuan, with plague outbreaks reported several times since the first confirmation of plague foci, among the two areas, animals plague mostly occurred in woodland, cultivation or bush while human plague occurred by uncertain reason, which all above have been caused great burden of local residents [8,9].
The Jianchuan plague focus was identified in 1974, until now animal plague was reported many times but no human cases reported. The main reservoir are Apodemus chevrieri and Eothenomys miletus, and Ctenophthalmus (Sinoctenophthalmus) quadrates and Neopsylla specialis specialis are as the major vector to transmit Y. pesti s [9]. The Yulong plague focus is located to the north of Jianchuan plague focus and is connected with the Jianchuan plague focus. On October 25, 2005, five cases of severe pneumonia plague and two deaths were reported among residents of the same village in Yulong County [8], later the plague focus was confirmed in Yulong county in 2006 by Y. pestis culture, but the composition and distribution of small mammals, fleas and microenvironment in this focal area is unclear until now [10]. On April, 2017, one small mammal (Rattus tanezumi) and two fleas (Neopsylla specialis specialis and Frontopsylla spadix spadix) were successfully isolated Y. pestis in Jianchuan focus implied the animal plague epidemic is ongoing and the outbreak of human plague may occur at any time in the focal area [9].
Comparing to Jianchuan plague focus, however, limited researches of small mammals and fleas in the Yulong plague focus may cause us lose the initiative to prevent and control plague before the disease occurred in human. Once the environment variables are changing, they will be affecting the abundance of small mammals and fleas and plague will be re-mergence in human again. Therefore, we need to make efforts concentrated on composition of small mammals and parasitic fleas in the Yulong plague focus, and comparing to Jianchuan plague to evaluated structure and characteristics of Yulong plague foci clearly, where is a new relatively active plague natural focus in Yunnan province.
Plague mainly depends on the animal hosts and vector fleas. The animal hosts parasitized by fleas plays an important role in plague transmission and conserving Y. pestis [11]. In a plague natural focus, one of the main approaches to controlling the incidence of the disease is by controlling the dynamics of host and flea populations [12]. However, previous study was mostly analyzed the characteristics of the Yulong focus from the Y. pestis [13]. It is still unclear whether the dominant small mammals and vector fleas are similar to the adjacent Jianchuan focus. In this study, we aim to investigate the distribution and composition of small mammals and parasitic fleas in the Yulong plague focus by comparing with those in the Jianchuan plague focus. This finding will be better understanding of the composition and distribution of small mammals and fleas may contribute to developing earlier warning methods for human plague outbreaks.

Study Design
This is a cross-sectional study based on the epidemiological information for the plague natural foci in the Yunnan province, two wild rodent plague foci included Yulong and Jianchuan were selected [8]. Small mammals were captured and parasitic fleas collected seasonally in these foci from December 2015 to October 2016.

Study Setting
The Yulong and Jianchuan plague foci are all located in the middle of the Hengduan Mountains with similar geographical environment, but separated by the low-lying valley in the center of the two regions. The Yulong plague focus is located in the Jianchuan plague focus is closing to Yulong focus (latitude, 26°10' N to 26°45' N, longitude 99°28' E to 99°56' E). The landscape is cultivation with pine forest mountain area between 2100-3000 meter, having a cold temperate climate [14]. Previously studies showed Yulong focus ever had human plagues with two people died but Jianchuan focus has long plague history only animals plagues reported [8,9,15].

Sampling Period
The sampling was separately conducted in each focus in each season. The four seasons are winter (which started from 30th Nov to 31 st Dec, 2015), spring (6 th Mar to 29 th Mar, 2016), summer (7 th July to 16 th July, 2016), autumn (9 th Oct to18 th Oct, 2016).

Collection of Small Mammals and Parasitic Fleas
In this study, the areas for wild trapping setting were selected  [16]. Then, small mammals were anesthetized by ether, the parasitic fleas on their body also were anesthetized and collected using a toothbrush to comb [17]. The fleas were stored in 75% ethanol for preservation and later identification under a light microscope based on the characteristic of fleas [18].

Statistical Analysis
Data were encoded and digitized using the EpiData software(3.1 version) and analyzed using the R software(4.0.2 version) [19].  [20,21]. A proportion of more than 10% was considered to indicate "dominant" species, 1% to 10% "common" species, and less than 1% "rare" species. The following international definitions of various parasitic indicators were adopted [22,23]: flea prevalence (%) = (number of fleainfected small mammals/total number of small mammals captured) ×100, and flea index = (total number of fleas/total number of small mammals captured). Commonly used Chinese definitions were used for the capture efficiency rate and small mammal density, as follows: capture efficiency (%) = (number of collected traps/number of traps placed)×100, small mammal density (%) = (number of small mammals captured/number of collected traps)×100 [24].

Results
In this present study, 10,120 traps were placed and 9,937 were recovered, with a capture efficiency rate of 98.20%. A total of 1953 small mammals were captured, the total small mammals captured density was 19.65%. The small mammals represented 24 species, 14 genera, 6 families, and 3 orders, of which 40.86% were Apodemus chevrieri, 25.40% were Eothenomys miletus, 14.50% were Apodemus draco, and 2.97% were Eothenomys proditor. In addition, 514 small mammals were infected by fleas, the total flea prevalence was 26.32%. Overall, 1102 parasitic fleas were collected, the total flea index was 0.56. The parasitic fleas represented 11 species, 7 genera, 3 families, and 6 subfamilies, of which 30.94% were Neopsylla specialis specialis, 37.48% were Ctenophthalmus (Sinoctenophthalmus) quadrates, 6.90% were Macrostylophora (M.) euteles, and 5.35% were Ctenophthalmus (Sinoctenophthalmus) yunnanus. The difference in capture efficiency rate between the Yulong and Jianchuan plague focus was not significant (Chi-squared test, P=0.1258). The small mammal density in the Jianchuan plague focus was significantly higher than that in the Yulong plague focus (Chi-squared test, P<0.01). Table 1     Note: a Four specimens of Soriculus leucopus in winter and one specimen of Vernaya fulva in summer.
b Two specimens of Eothenomys melanogaster and one specimen of Rattus rattus/sladeni in summer; one specimen of Deficiency in winter.      (Table 5). Niviventer Tupaia be-

Discussion
In this study, we analyzed the composition and distribution of focus. The main flea difference in the two foci, however, which may be caused by their independent ecological circle [9,25]. This also implied that Yulong focus has a different composition of small mammals and fleas even adjacent to Jianchuan focus, further we need to effort long-term monitoring data to determine whether this composition is accidental or long-term, which to confirm whether impact occurrence of plague. are consisted with previously studies, A. chevrieri and E. miletus are dominant species of small mamamls in the two focal areas [26].
Additionally, it is worth noting that A. draco was the dominant species, the captured number was even more than those in E. miletus in Yulong focus, previously studies showed that A. draco is an invasive species but E. miletus is the native species in the middle of the Hengduan Mountains, in order to adapt and survive, A. draco needs to constantly adjust its physiological characteristics such as basal metabolic rate [27].
In this study, the mostly increased number of A. draco shows maybe its further adapted well to the geographical climate of the Hengduan Mountains, but whether the increase in number will increase the occurrence of animal plague requires long-term monitoring and judgment. Furthermore, E. proditor was captured in the Yulong plague focus but not in the Jianchuan plague focus, it might be a secondary host or a popular host, but not be as  [44,45]. The evidence of a link between meteorological variables, small mammal density, and risk of infection outbreaks is compelling [46]. This present study has certain strength. First of all, we compared the composition and seasonal distribution of small mammals and parasitic fleas in two similar and adjacent wild rodent foci, but found that although the two places are similar, the existence of species in each season is obviously different. This may be due to the two place is separated by a canyon geographically, which has led to climate change, but further research is still needed. Secondly, our research is different from previous studies on plague foci. We focus more on observing the population of small mammals and fleas in the four seasons, and we can find the number and changes of small mammals and fleas in different seasons. This also provides a scientific basis for local government to effectively implement small mammals and flea control. We also have certain limitations, however, because we are a cross-sectional study, we cannot prove the causality. Secondly, we have only investigated for a year, and the conclusions we got need more time to monitor to be corroborated.
This time we only roughly divided into four seasons to collect small mammals and parasitic fleas, but did not carefully analyze the differences in temperature, humidity, and food in each season.
These may affect the composition of small mammals and fleas. Our team next step will consider take these factors into further analysis.

Conclusion
The

Ethics Statement
The study has been approved the Medical Ethics Committee

Consent for publication
Not available.

Availability of Data and Materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.