EcoHealth

, Volume 7, Issue 1, pp 18–23

Broad Distribution of Ranavirus in Free-Ranging Rana dybowskii in Heilongjiang, China

  • Kai Xu
  • Dong-Ze Zhu
  • Ying Wei
  • Lisa M. Schloegel
  • Xiao-Feng Chen
  • Xiao-Long Wang
Short Communication

DOI: 10.1007/s10393-010-0289-y

Cite this article as:
Xu, K., Zhu, DZ., Wei, Y. et al. EcoHealth (2010) 7: 18. doi:10.1007/s10393-010-0289-y
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Abstract

Ranaviruses have been associated with die-offs in cultured amphibians in China, but their presence in wild amphibians has not yet been assessed. We sampled free-ranging Rana dybowskii at seven sites throughout Heilongjiang Province to determine the presence and prevalence of ranaviruses in this region. Our results revealed an overall infection prevalence of 5.7% (18/315) for adults and 42.5% (51/120) for tadpoles by PCR. PCR-amplified product showed a high degree of homology with several members of the Iridoviridae, mostly with those belonging to the genus Ranavirus. The results indicate that ranaviruses are broadly distributed throughout Heilongjiang Province and could have important implications for the health of native wildlife. Additional sampling and management strategies should be urgently adopted to address the prevalence and health consequences of ranaviruses throughout China.

Keywords

ranavirusRana dybowskiifree-ranging populationdistributionnortheastern China

Introduction

Amphibian populations are experiencing drastic declines on a global scale (Stuart et al., 2004). Numerous factors have been implicated in the cause of these declines, including overharvesting and habitat destruction. Enigmatic events seem to be accountable for the majority of declines in recent times, such as disease emergence and spread (Daszak et al., 2003; Stuart et al., 2004; Lips et al., 2006; Rachowicz et al., 2006). Ranaviruses, family Iridoviridae, are of particular concern and so far have been linked to amphibian mortality events on five continents, including Europe, North America, Australia, South America, and Asia (Zupanovic et al., 1998; Zhang et al., 2001; Green et al., 2002; Jancovich et al., 2003; Galli et al., 2006; Miller et al., 2007).

Ranaviruses are known to infect a wide range of hosts, including fish, amphibians, and reptiles (Daszak et al., 1999). First isolated from the Northern leopard frog (Rana pipiens), ranaviruses have since been identified in numerous other anurans (Chinchar, 2002). Although the mechanism of ranavirus transmission remains unclear, both vertical and horizontal transmissions have been documented (Brunner et al., 2004; Pearman et al., 2004). Recent studies indicate the prevalence of ranaviruses is related to temperature, suggesting seasonal fluctuations of pathogen infection (Rojas et al., 2005) and distribution (Harp and Petranka, 2006; Greer and Collins, 2008). A number of authors also provide evidences linking the anthropogenic movement of animals to the occurrence of ranaviruses in many regions (Go et al., 2006; Pasmans et al., 2008; St-Amour et al., 2008; Schloegel et al., 2009).

There have been a number of reported cases of ranavirus associated mortality events in Asia, including die-offs of the cultured tiger frog (Rana tigrina) in Thailand (Kanchanakhan, 1998). In China, reported outbreaks and deaths have been attributed to Rana grylio virus (RGV) in cultured pig frogs (Rana grylio) in Hunan and Hubei Provinces, and tiger frog virus (TFV) in cultured tiger frogs (Rana tigrina rugulosa) in Guangdong Province (Zhang et al., 2001; He et al., 2002; Weng et al., 2002). To date, there have been no reports of ranavirus infections in local, free ranging amphibians in China. Due to the pathogenic nature of infection with RGV and TGV in cultured frogs, the risk to native, wild amphibians could be significant.

Dybowski’s frog (Rana dybowskii) is a typical forest inhabitant in the mountain ranges of northeastern China. Populations of Dybowski’s frog have experienced pressure from numerous sources in recent years that could predispose them to disease. During the past two decades, the harvesting of these animals for consumptive and scientific purposes has increased. Such human disturbances to amphibian populations often can trigger a stress response in animals, which could potentially increase susceptibility to disease through immunosuppression (Carey, 1993; Carey et al., 1999). Furthermore, the cultivation of live, North American bullfrogs (Lithobates catesbeiana) in Asia is increasing. North American bullfrogs are known to be carrier hosts of ranaviruses in the wild and the live animal trade (Gray et al., 2007; Mazzoni et al., 2009; Schloegel et al., 2009). The increasing bullfrog trade could serve to increase the risk of pathogen introduction and spread to wild populations through the escape of infected individuals.

To determine the presence and prevalence of ranaviruses in wild amphibians in China, we used R. dybowskii as our study species because of its vulnerable status and declining numbers (Zhao, 1998; Liu et al., 2007). This is the first survey of parasitosis by ranavirus in wild amphibians in China.

Materials and Methods

Field Sampling for Adult Frogs

We conducted sampling surveys of Rana dybowskii for ranaviruses across seven regions (3 sites/region) in Heilongjiang Province: (1) Attached Forestry Farms, Hebei Forest Bureau, Hebei town (sites A1–A3); (2) Attached Forestry Farms, Dongfanghong Forestry Bureau, Dongfanghong town (sites B1–B3); (3) Provincial Nature Reserve, Acheng city (sites C1–C3); (4) Attached Forestry Farms, Heihe Forestry Bureau, Heihe city (sites D1–D3); (5) Forestry Farms, Tieli city (sites E1–E3); (6) Attached Forestry Farms, Huanan Forestry Bureau, Huanan county (sites F1–F3); and (7) Attached Forestry Farms, Hailin Forestry Bureau, Mudanjiang city (sites G1–G3). The seven regions combined encompass the majority distribution of Rana dybowskii in Heilongjiang. Within each region, the three study sites were distanced 3 km from each other and bordered the banks of streams or ponds. In each sampling site, 15 samples were randomly collected (Fig. 1). A total of 315 adult frogs were sampled (Table 1). Frogs were collected from after hibernation in the spring. Frogs were captured using a hand-net, placed in disposable plastic bags and then transported back to the laboratory. Animals were kept individually and a fresh pair of gloves was used when handling each animal to avoid cross-contamination. All animals were anesthetized in accordance with laboratory protocols and a piece of the liver tissue was biopsied.
https://static-content.springer.com/image/art%3A10.1007%2Fs10393-010-0289-y/MediaObjects/10393_2010_289_Fig1_HTML.gif
Figure 1

Map and location of sampling regions in Heilongjiang Province, China.

Table 1

Number of R. dybowskii Positive for Rv by PCR by Region and Site

Region

Site ID and locate

Adults % infected by site (N)

Tadpoles % infected by site (N)

Adults % infected by region (N)

Tadpoles % infected by region (N)

1. Hebei

A1: N47°59′45″, E130°06′28″

13.3 (15)

80 (10)

4.4 (45)

66.7 (30)

A2: N47°52′51″, E130°10′36″

0 (15)

70 (10)

  

A3: N47°39′58″, E130°18′22″

0 (15)

50 (10)

  

2. Dongfanghong

B1: N46°24′11″, E133°26′38″

6.7 (15)

30 (10)

6.7 (45)

23.3 (30)

B2: N46°21′15″, E133°30′12″

0 (15)

0 (10)

  

B3: N46°23′15″, E133°29′12″

13.3 (15)

40 (10)

  

3. Acheng

C1: N45°28′49″, E127°35′36″

0 (15)

0 (10)

0 (45)

0 (30)

C2: N45°28′54″, E127°33′52″

0 (15)

0 (10)

  

C3: N45°27′10″, E127°31′55″

0 (15)

0 (10)

  

4. Heihe

D1: N49°51′19″, E127°11′04″

13.3 (15)

90 (10)

8.9 (45)

80 (30)

D2: N49°51′28″, E127°02′15″

13.3 (15)

90 (10)

  

D3: N49°52′07″, E127°04′07″

0 (15)

60 (10)

  

5. Tieli

E1: N47°17′35″, E128°23′04″

6.7 (15)

4.4 (45)

-

E2: N47°18′01″, E128°25′47″

6.7 (15)

  

E3: N47°16′10″, E128°24′52″

0 (15)

  

6. Huanan

F1: N46°12′47″, E130°38′25″

6.7 (15)

4.4 (45)

F2: N46°13′03″, E130°37′59″

0 (15)

  

F3: N46°12′24″, E130°39′06″

6.7 (15)

  

7. Hailin

G1: N44°33′32″, E129°15′48″

13.3 (15)

11.1 (45)

G2: N44°33′49″, E129°16′34″

13.3 (15)

  

G3: N44°32′44″, E129°16′01″

6.7 (15)

  

Data are percentages with numbers in parentheses unless otherwise indicated.

Field Sampling for Larvae

A total of 120 larvae (10/site) were collected during the early summer from calm streams and natural ponds in the regions of Hebei, Dongfanghong, Acheng, and Heihe. Water bodies were located approximately 3 km from each other in relatively pristine environments (Table 1).

PCR Assay

Viral DNA from the liver of adult specimens was isolated using the standard TIANamp Genomic DNA Kit Protocol for Animal Tissue (TIANGEN Co. Ltd., China). Viral DNA of larvae was extracted from the homogenate of the larvae’s body minus the mouthparts and tail. TIANamp Genomic DNA Kit for Animal Tissue (TIANGEN Co. Ltd., China) also was used.

PCR Amplification was conducted using primers specific for a 500 bp fragment of the ranavirus major capsid protein (MCP) (Mao et al., 1997) in 20 μl PCR reactions. The kit of 2× Taq PCR MasterMix (TIANGEN Co. Ltd., China) was used for PCR reactions: template 0.8 μl (0.2 μg), ddH2O 8.4 μl, 2× PCR MasterMix 10 μl and each 0.4 μl (20 μM) for forward and reverse primer. Thermocycling conditions were identical for all samples: 94°C 5 min; 94°C 30 s, 55°C 30 s, and 72°C 30 s, 35 cycles followed by an extension of 72°C 2 min.

Five positive amplicons were selected randomly for sequencing and were compared to previously published sequences available in GenBank using the BLAST search tool in the NCBI website.

Results

Adults

Individuals collected from six of seven sample regions (Hebei, Dongfanghong, Heihe, Tieli, Huanan, and Hailin) were found to be PCR-positive for ranaviruses. Uninfected sites, however, were present in each region (Table 1). The prevalence of infection in each of the six Rv-positive districts was: 4.4, 6.7, 8.9, 4.4, 4.4, and 11.1%, respectively. Acheng city (Region 3) was the only region found to be negative for ranavirus by PCR. The overall prevalence of ranavirus in adult Rana dybowskii in Heilongjiang was 5.7% (Table 1). Individuals sampled did not exhibit any observable signs of infection.

Tadpoles

Larvae from three of four sample regions (Hebei, Dongfanghong, and Heihe) were Rv-positive by PCR with prevalence rates of 66.7, 23.3, and 80%, respectively. In accordance with the results from adult specimens, Acheng (Region 3) was the only region to yield negative results in 100% of individuals sampled. The overall prevalence of infection for tadpoles was 42.5% (Table 1).

Primary Viral Identification

Analyses of amplified MCP sequences from the five selected samples showed a 98% homology with Iridovirus RGV-9806 MCP (AF192509), Frog Virus 3 complete genome (AY548484), Frog Virus 3 viral core protein (U36913), Iridovirus RGV-9807 MCP (AF192508), Terrapene Carolina ranavirus MCP (U82553), and Bohle Iridovirus MCP (AY187046).

Discussion and Conclusions

Our study is the first to definitively identify the presence of ranaviruses in wild anurans in China. Positive results indicate that ranaviruses maintain a broad distribution throughout Heilongjiang Province, with the exception of a few scattered regions that appear to be virus-free (Table 1). Overall prevalence of infection in adults was low (5.7%); however, infection in tadpoles was much greater (42.5%). Previous studies also have reported a greater susceptibility of infection in amphibian larvae compared with adults (Gantress et al., 2003; Brunner et al., 2005).

The prevalence of ranavirus infection across the 21 sample sites varied greatly (0–90%). Local environmental conditions, including forest coverage, human disturbances, pesticide and herbicide use, and climatic and geographical conditions were recorded and briefly analyzed to determine whether they had an impact on virus prevalence. Initial estimations were rough and more precise data are required to adequately assess their impact in future studies.

Numerous authors have implicated the live animal trade in the spread of amphibian pathogens, including ranaviruses (Picco and Collins, 2008; Schloegel et al., 2009). A recent study of farmed North American bullfrogs (Lithobates catesbeiana) in Brazil revealed that trade isolates of the fungal pathogen Batrachochytrium dendrobatidis are genetically similar, and in some cases identical, to isolates from native Latin American amphibians (Schloegel et al., 2010). These data suggest that farmed frogs may act as reservoirs for the spread and persistence of amphibian pathogens. Ranaviruses have already been documented in cultured frogs in Asia (Zhang et al., 2001; Weng et al., 2002). Comparative molecular analyses of ranavirus infections in the trade versus those in the wild could help to determine whether frog farms in China are contributing to infection prevalences in native wildlife.

Studies of Ambystoma tigrinum virus (ATV) indicate that, although infected larval deaths in amphibians increase with increasing density, the virus does not appear to drive infected populations to extinction (Greer et al., 2008). Environmental reservoirs and additional hosts (e.g., L. catesbeiana), however, could alter disease dynamics, particularly in the presence of additional pathogens (e.g., Batrachochytrium dendrobatidis). B. dendrobatidis is thought to have played a role in the declines and extinctions of numerous amphibians globally (Schloegel et al., 2006). Studies are beginning to identify the presence of this pathogen in amphibian populations in Asia (Rowley et al., 2007; Kusrini et al., 2008; McLeod et al., 2008; Une et al., 2008; Wei et al., 2010). Further studies are required to understand the host pathogen dynamics at play in China and whether native amphibians are at risk of death and decline and even extinction.

Viral presence in frog populations has been shown to be inconsistent from year to year (Brunner et al., 2004). Long-term monitoring is therefore essential in this district for evaluating the frequency and reoccurrence of viral infections. Ranaviruses also are known to cause disease across a range of animals (i.e., amphibians, fish, and reptiles; Daszak et al., 1999). Additional surveys for the presence of ranavirus in Chinese wildlife are important for evaluating infection prevalence and spread throughout the region.

This study is the first report of viruses from the family Iridoviridae in free-ranging amphibian populations in China. The data that we obtained are important for assessing the health and conservation status of native amphibian fauna in the face of constantly changing environmental factors. Our results significantly expand our scope of the epidemiology of ranavirus in China and highlight the necessity for the collaboration of local and international scientists to address the risks to the health and survival of native, Chinese wildlife.

Acknowledgments

Supported by China Postdoctoral Science Foundation (Code: 20080430872).

Copyright information

© International Association for Ecology and Health 2010

Authors and Affiliations

  • Kai Xu
    • 1
  • Dong-Ze Zhu
    • 1
  • Ying Wei
    • 1
  • Lisa M. Schloegel
    • 2
  • Xiao-Feng Chen
    • 1
  • Xiao-Long Wang
    • 1
  1. 1.Wildlife Resource CollegeNortheast Forestry UniversityHarbinPeople’s Republic of China
  2. 2.Wildlife TrustNew YorkUSA