Abstract
Background:
Rodent is a reservoir of various zoonotic pathogens. Wanzhou section of the Three Gorges reservoir region (TGRR) is a superior habitat for rodents, and the situation of rodent-borne zoonotic pathogens in this region has not been surveyed in recent years.
Materials and Methods:
Rodents were night trapped with mousecage or mousetrap in urban and surrounding towns’ indoor or outdoor areas of the Wanzhou section of the TGRR, and nucleic acid was extracted from their lung or a mixture of liver, spleen, and kidney. Commercialized qPCR kits for pathogenic Leptospira spp., Rickettsia typhi, Anaplasma phagocytophilum, Bartonella spp., Orientia tsutsugamushi, and Francisella tularensis and qRT-PCR kits for hantavirus (HV), and severe fever with thrombocytopenia syndrome virus (SFTSV) were used for the detection of associated pathogens in collected rodents.
Results:
From 2021 to 2023, 604 rodents belonging to 10 species were collected. HV and pathogenic L. spp. were detected positive, with infection rates of 0.66% (4/604) and 1.32% (8/604), respectively. B. spp. were detected positive with an infection rate of 4.73% (19/402) in the rodents trapped in 2022 and 2023. Other five pathogens were all detected negative.
Conclusion:
This study showed that the Wanzhou section of the TGRR had HV, pathogenic L. spp., and B. spp. co-circulation in rodents. Hence, more attention should be paid to the prevention and control of associated rodent-borne diseases.
Introduction
Rodents are distributed almost worldwide and act as a reservoir of various zoonotic pathogens (Capizzi et al, 2014; Han et al, 2015; Meerburg et al, 2009). Rodents could carry pathogens of at least 60 zoonotic diseases, including viruses, bacteria, rickettsia, spirochetes, and parasites, and play a major role in their transmission in different ways (Dahmana et al, 2020; Han et al, 2015; Meerburg et al, 2009). Among these pathogens, pathogenic Leptospira spp., Rickettsia typhi, Anaplasma phagocytophilum, Bartonella spp., Orientia tsutsugamushi, Francisella tularensis, hantavirus (HV), and severe fever with thrombocytopenia syndrome virus (SFTSV) are important in terms of public health in China (Jian et al, 2022; Liu et al, 2018; Liu and Zhang, 2023; Luo et al, 2023; Meerburg et al, 2009; Song et al, 2023; Sun et al, 2023; Xu et al, 2022; Zhang et al, 2019; Zhang et al, 2021).
Wanzhou District is part of Chongqing City and is located in the center zone of the Three Gorges reservoir region (TGRR), which has suitable environment for rodents. Previous studies indicated that pathogenic L. spp. and HV were co-circulated in rodents in TGRR from 1997 to 2012 and corresponding diseases were decreased (Bai et al, 2015; Chang et al, 2016). However, to our knowledge, the situation of rodent-borne zoonotic pathogens in the TGRR has not been surveyed in recent years.
In this study, we collected rodents in Wanzhou District from 2021 to 2023 and investigated the situation of pathogenic L. spp., R. typhi, A. phagocytophilum, B. spp., O. tsutsugamushi, F. tularensis, HV, and SFTSV in these rodents.
Materials and Methods
Ethics statement
No permission was required for the described studies because no endangered or protected species were involved. Rodent collection was approved by the owners at each location, and all collected rodents were euthanized in strict accordance with the Guidance for Experimental Animal Welfare and Ethical Treatment by the Ministry of Science and Technology of China.
Sample collection
From 2021 to 2023, rodents were night trapped with mousecage or mousetrap in urban and surrounding towns’ indoor or outdoor areas of Wanzhou District (Fig. 1) every odd-numbered month, referring to the period from November of the previous year to September of the current year. The captured rodents were euthanized, identified of species, and then aseptically dissected. Lung, liver, spleen, and kidney were collected in sterile 2 mL tube and stored at −80°C until nucleic acid extraction.
Geographic map showing the location of sampling spots in the Wanzhou section of the TGRR.
Nucleic acid extraction
Twenty-five milligrams of lung or a mixture of liver, spleen, and kidney was mixed with 500 μL
Rodent-borne zoonotic pathogen detection
Commercialized qPCR kits for pathogenic L. spp., R. typhi, A. phagocytophilum, B. spp., O. tsutsugamushi, and F. tularensis were purchased from Mabsky (Shenzhen, China), and qRT-PCR kits for HV and SFTSV were purchased from Bioperfectus. The nucleic acid extracts of lung were used for HV detection, and the nucleic acid extracts of the mixture of liver, spleen, and kidney were used for the detection of other pathogens. Samples collected in 2021 were just used for the detection of pathogenic L. spp., R. typhi, HV, and SFTSV. Samples collected in 2022 and 2023 were used for the detection of all eight pathogens.
The qPCR/qRT-PCR amplification was carried out in a CFX96 Real-Time system (Bio-Rad, CA, USA) according to the manufacturer’s instructions. Detection of pathogenic L. spp., R. typhi, A. phagocytophilum, B. spp., O. tsutsugamushi, and F. tularensis used the following thermal profile: Incubation at 50°C for 2 min for UNG action (eliminating PCR amplicons’ contaminant), then an activation step at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 5 s, and an annealing–extension at 55°C for 60 s combined with fluorescence acquisition. Detection of HV and SFTSV used the following thermal profile: incubation at 50°C for 10 min for reverse transcription, then an activation step at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 10 s, and an annealing–extension at 58°C for 30 s combined with fluorescence acquisition. Positive and negative controls were set for every test by using plasmids carrying target sequence of the corresponding pathogen and ddH2O, respectively.
Results
From 2021 to 2023, 604 rodents were trapped in urban and surrounding town areas of Wanzhou District. The species identification of 604 rodents was done based on morphology, resulting in the identification of Rattus flavipectus (44.20%), R. norvegicus (22.68%), Anourosorex squamipes (22.52%), Mus musculus (2.15%), R. edwardsi (1.99%), Niviventer confucianus (1.99%), Apodemus agrarius (1.66%), R. nitidus (1.16%), Crocidura attenuata (0.99%), and Sorex araneus (0.66%). All 10 species of rodents were trapped in 2021, whereas S. araneus and R. nitidus were not trapped in 2022 and 2023, respectively (Table 1).
Rodents Captured in Wanzhou District and Their Infection Rates of Rodent-Borne Zoonotic Pathogens, 2021–2023
Rodents detected positive of rodent-borne pathogens were displayed in bold. Rickettsia typhi, Anaplasma phagocytophilum, Orientia tsutsugamushi, Francisella tularensis, and SFTSV were not listed because no positive.
HV, hantavirus.
HV was detected positive, with an infection rate of 0.66% (4/604), and its infection rates of 2021 and 2023 were 0.99% (2/202) and 1.00% (2/201), respectively, whereas it was not detected in 2022. Rodents infected with HV were all R. flavipectus and R. norvegicus, and the infection rate of R. norvegicus (2.19%) was higher than R. flavipectus (0.37%) (Table 1).
Pathogenic L. spp. were detected positive, with an infection rate of 1.32% (8/604), and their infection rates from 2021 to 2023 were 2.49% (5/202), 0.50% (1/201), and 1.00% (2/201), respectively. Three R. flavipectus and five R. norvegicus were positive of pathogenic L. spp. with a higher prevalence observed in R. norvegicus (3.65%) compared with R. flavipectus (1.12%) (Table 1). Furthermore, co-infection of HV and pathogenic L. spp. was found in one R. flavipectus and two R. norvegicus.
B. spp. were detected positive, with an infection rate of 4.73% (19/402), and their infection rates in 2022 and 2023 were 3.98% (8/201) and 5.47% (11/201), respectively. In the 10 species of trapped rodents, B. spp. were detected in six species, including R. flavipectus, R. norvegicus, R. edwardsi, N. confucianus, A. agrarius, and C. attenuata, with infection rates of 6.21%, 3.13%, 18.18%, 27.27%, 12.50%, and 20.00%, respectively (Table 1). Besides these three pathogens, R. typhi, A. phagocytophilum, O. tsutsugamushi, F. tularensis, and SFTSV were all detected negative.
Discussion
In this study, 10 species of rodents from the orders Rodentia and Insectivora were trapped in Wanzhou District between 2021 and 2023, and the main species of these two orders were R. flavipectus and R. norvegicus and A. squamipes, respectively. Compared with the rodent species of Chongqing from 1997 to 2012, the number of species was increased, whereas constituent ratios of M. musculus and A. agrarius were declined (Chang et al, 2016). This is consistent with HE et al.’s study, which showed that constituent ratios of M. musculus and A. agrarius in the Chongqing section of the TGRR were declined after 2014 (HE et al, 2020). This population reshape of rodents may be the result of changes in living environment caused by water storage in the TGRR (Wan et al, 2022).
The infection rate of HV (0.66%) declined between 2021 and 2023, whereas that of pathogenic L. spp. (1.32%) was increased when compared with this of Chongqing from 1997 to 2012 (Chang et al, 2016). Moreover, the positive rate of pathogenic L. spp. in rodents in Hubei in 2021 (4.8%) and the positive rate of HV in Zhejiang in 2020 (1.86%) were both higher than those found in this study (Wu et al, 2022; Xu et al, 2022). This indicates timely and regional differences in the prevalence of HV and pathogenic L. spp., with pathogens exhibiting relatively low prevalence in rodents in the Wanzhou section of the TGRR. And the low prevalence of HV and pathogenic L. spp. would be the result of continuous rodent collection in a small area, which would restrict the spread and naturally reduce the prevalence of pathogens. In addition, co-infection of HV and pathogenic L. spp. was found in R. flavipectus and R. norvegicus in this study. This is consistent with previous studies, which showed that co-infection of different pathogens in rodents is universal (Costa et al, 2014; Jeske et al, 2021; Tadin et al, 2012).
The infection rate of B. spp. between 2022 and 2023 (4.73%) was lower, whereas the number of infected species was more than that in Handan City (28.4%) and Chengde City (6.7%) in 2020 (Jian et al, 2022). Our study showed that B. spp. could infect a great diversity of rodents, and this is consistent with previous studies (Krugel et al, 2022; Tian et al, 2023). The result, that A. phagocytophilum, O. tsutsugamushi, F. tularensis, and SFTSV were all detected negative, is consistent with the previous study, which showed that Chongqing City had no corresponding disease of these four pathogens (XU et al, 2022). To our knowledge, murine typhus, the disease caused by R. typhi, has rarely occurred in Chongqing City. This maybe the reason why R. typhi detected negative, but the accurate reason needs further research.
Conclusions
Taken together, this study shows that HV, pathogenic L. spp., and B. spp. were co-circulated in rodents in the Wanzhou section of the TGRR between 2021 and 2023. The prevention and control of associated rodent-borne diseases should be strengthened. And further studies should broaden the spectrum of detection of rodent-borne pathogens and combine with sequencing and more optimized rodent collection plan, so as to get more comprehensive and unbiased information about the prevalence situation of rodent-borne zoonotic pathogens in the Wanzhou section of the TGRR.
Footnotes
Acknowledgements
We are sincerely grateful to the Department of Disinfection and Vector Control staff of Chongqing Center for Disease Control and Prevention for their confirmation of rodent species same to this study based on molecular methods in the work of vector surveillance network in China.
Author’s Contributions
Sample collection: Yadong Tan, Jiang Wan, Xuewu Xiong and Jun Wang. Pathogen detection: Lei Zuo, Hengqin Wang, Wenli Tan and Yulu Gan. Data curation: Hengqin Wang, Yadong Tan and Jiang Wan. Writing and revision: Lei Zuo. Funding acquisition: Lei Zuo and Chao Luo.
Author Disclosure Statement
We declare that we have no conflict of interest.
Funding Information
This research was funded by the Key Project for Technological Innovation and Application Development of Chongqing (CSTB2022TIAD-ZXX0031) and the First Batch of Key Disciplines on Public Health in Chongqing.