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Soto-Azat Rv A SURVEILLANCE STUDY LINKS THE AFRICAN CLAWED FROG WITH THE INTRODUCTION OF RANAVIRUS TO CHILE Alexandra Peñafiel-Ricaurte, Stephen J. Price, Mario Alvarado-Rybak, Andrew A. Cunningham, and Claudio Soto-Azat IPBES report 2019 50% González del Pliego et al. 2019. Curr. Biol. • Plastics ↑10 times since 1980. • Population x2 since 1970. • ↓82% biomass wild mammals. INTRODUCED SPECIES MAY PLAY AN IMPORTANT ROLE IN RANAVIRUS EPIDEMIOLOGY • There is evidence that X. laevis and different species of introduced fish may act as Ranavirus reservoirs or vectors for native amphibian species (Gantress et al. 2003, Robert et al. 2007, Bang Jensen et al. 2009, Gobbo et al. 2010, Bang Jensen et al. 2011, Brenes et al. 2014, Chinchar et al. 2014). • In Chile: Mosquito fish X. laevis • 26 introduced fish spp. have established feral populations in rivers and other water bodies in Chile (Iriarte et al. 2005). Global distribution of Xenopus laevis Página 1 | El Diario de Atacama 03-06-19 06(47 http://www.diarioatacama.cl/impresa/2019/06/02/full/cuerpo-principal/1/ Página 1 de 2 Mora et al. 2019. Manag. Biol. Inv. Non-linear X(t)=N(1)(1-exp(-Kt)) extrapolation model • Captured individuals = average of 218.4 individuals/hour. • Population size: 1,162 individuals. • Divided by area (86 m2): density of 13.5 individuals/m2. RANAVIRUS HAS BEEN REPORTED IN SEVERAL COUNTRIES FROM SOUTH AMERICA FIRST EVIDENCE OF RANAVIRUS IN CHILEAN AMPHIBIANS WAS REPORTED IN 2016 • 2011 – 2013. Calyptocephalella gayi Pleurodema thaul 9 individuals 2 individuals Telmatubofo bullocki Xenopus laevis 1 individual 175 individuals TOTAL: 187 individuals Soto-Azat et al. 2016 RANAVIRUS WAS DETECTED IN ONE NATIVE AND 7 INVASIVE AMPHIBIANS FROM TWO SITES IN SANTIAGO Site 1: Rinconada (4 X. laevis). Site 3: Talagante (3 X. laevis, 1 Calyptocephalella gayi). 4.3% FV3 RANAVIRUS SURVEILLANCE ALONG CHILE • Tadpoles from native non-endangered species. • Rhinella spinulosa. (98 tadpoles, II region) • Pleurodema thaul. (335 tadpoles, IV, V, VI, VII, XIV region). • Calyptocephalella gayi. (5 dead tadpoles, IV, VII región) (58 preserved carcasses, RM). TOTAL: 1752 Amphibians • Xenopus laevis. 741 Native + 1011 X. laevis (1011 post-metamorphs, IV, V, VI, VII región). • Adult amphibians from native species. (245 individuals from 11 species II, IV, VIII, X región). FISH SURVEILLANCE Invasive freshwater fish • Gambusia holbrooki. (321 adults, II, VI, V, VI, XIV). • Oncorhynchus mykiss. (71 juveniles, II, X region). • Cyprinus carpio. (3 adults, VIII region). Native freshwater fish • Orestias chungarensis. (30 adults, XV region). TOTAL: 526 fish • Cheirodon galusdae. 395 Invasive + 101 native (31 adults, VIII region). • Galaxia maculatus. (70 adults, VIII region). SAMPLING Adult amphibians from native Native tadpoles, X. laevis and fish species • Euthanasia (MS-222) (Landsberg et al. • Swab tips preserved in 95% ethanol. 2013; Bayley et al. 2013). • Liver, kidney and spleen, preserved at -80°C. • qPCR assay specific for amphibian-associated ranaviruses, targeting a 97bp fragment of the MCP gene (Leung et al. 2017). • 84bp fragment of the EBF3 gene (common from vertebrates) for viral load estimation. • Also used as an IPC to discard PCR inhibition. RESULTS: AMPHIBIAN TISSUE SAMPLES SUMMARY True Region Site Lat Long Species n Rv + Prevalence Lower Upper VX Putre -18.195503 -69.568573 Rhinella spinulosa 54 0 0% 0 0 6.641359 II Alcolcha -21.027084 -68.450103 Rhinella spinulosa 32 0 0 0 10.71792 Valle de Jere -23.187166 -67.991194 Rhinella spinulosa 5 0 0% 0 0 43.448246 Ca l a ma -22.748641 -68.071030 Rhinella spinulosa 7 0 0 0 35.433044 IV Rio Elqui -29.897250 -71.244583 Pleurodema thaul 81 0 0 0 4.527809 Ovalle -30.644806 -71.568204 Xenopus laevis 48 1 2.0833333 0.10686103 10.899218 Illapel -31.619583 -71.141833 Pleurodema thaul 23 0 0 0 14.311662 Calyptocephalella gayi 2 0 0 0 65.761977 V Jardin Botanico -32.039694 -71.498111 Xenopus laevis 39 0 0 0 8.966685 Villa Alemana -33.036251 -71.370742 Xenopus laevis 285 1 0.3508772 0.01799765 1.96041 -34.185358 -70.799575 Pleurodema thaul 58 0 0 0 6.211786 MR Criadero rana chilena -33.385197 -71.645147 Calyptocephalella gayi 58 0 0 0 6.211786 Talagante -33.686389 -70.908333 Xenopus laevis 211 1 0.4739336 0.02430962 2.635253 Rinconada de Maipu -33.496111 -70.829722 Xenopus laevis 297 6 2.020202 0.92908604 4.336631 VI Rancagua -34.185358 -70.799575 Pleurodema thaul 59 0 0% 0 0 6.112937 -34.168433 -70.854356 Xenopus laevis 60 0 0 0 6.017185 VII Hualañé -34.974352 -71.850175 Xenopus laevis 71 0 0 0 5.132795 Calyptocephalella gayi 3 0 0% 0 0 56.149703 Río Mataquito -35.045768 -71.737533 Pleurodema thaul 57 0 0 0 6.313883 XIV Valdivia -39.872720 -73.160637 Pleurodema thaul 57 0 0% 0 0 6.313883 TOTAL 1507 9 • Overall apparent prevalence in X. laevis: 0.9% (9/ 1011) LOW VIRAL LOADS WERE DETECTED IN ALL POSITIVE SAMPLES Region Study Site Sample ID # Viral copies IV - Coquimbo Ovalle MUR12/17 3.01 V - Valparaiso Villa Alemana VA20/16 8.11 Metropolitan Talagante ETA29/17 3.52 Metropolitan Rinconada ERI36/17 2.71 Metropolitan Rinconada ERI49/17 4.06 Metropolitan Rinconada MRI04/17 2.61 Metropolitan Rinconada MRI13/17 8.7 Metropolitan Rinconada MRI15/17 6.57 Metropolitan Rinconada MRI27/17 4.61 Field signs and lesions absence Carrier - Reservoir Risk for native amphibian species RESULTS: ADULT NATIVE AMPHIBIANS SAMPLES SUMMARY NATIVE AMPHIBIANS NON-INVASIVE SAMPLES SUMMARY Apparent Region Site Lat Long Species n Rv + Prevalence II Rio Loa -22.748641 -68.071030 Telmatobius dankoi 50 0 0 Ca l a ma -22.748641 -68.071030 Rhinella spinulosa 18 0 0 Telmatobius vilamensis 10 0 0 Valle de Jere -23.187166 -67.991194 Rhinella spinulosa 30 0 0 IV Rio Elqui -29.897250 -71.244583 Pleurodema thaul 51 0 0 Calyptocephalella gayi 5 0 0 VIII Nahuelbuta -37.882251 -73.371236 Batrachyla taeniata 0%4 0 0 Calyptocephalella gayi 11 0 0 Rhinoderma darwinii 8 0 0 Eusophus contulmoensis 11 0 0 Pleurodema thaul 14 0 0 X Puyehue -40.723249 -72.433281 Rhinoderma darwinii 7 0 0 Eusophus calcaratus 23 0 0 Eusophus vertebralis 2 0 0 Eusophus emiliopigini 1 0 0 TOTAL 245 0 RESULTS: FRESHWATER FISH SAMPLES SUMMARY FRESHWATER FISH SAMPLES SUMMARY (TISSUE) Apparent Region Site Lat Long Species n Rv + Prevalence XV Lago Chungará -18.235944 -69.181638 Orestias chungarensis 30 0 0 II Ca l a ma -22.501987 -68.960164 Gambusia holbrooki 70 0 0 Oncorhynchus mykiss 11 0 0 IV Rio Elqui -29.897250 -71.244583 Gambusia holbrooki 82 0 0 V Jardin Botanico -32.039694 -71.498111 Gambusia holbrooki 40 0 0 Villa Alemana -33.036251 -71.370742 Gambusia holbrooki 50% 0 0 VI Rancagua -34.185358 -70.799575 Gambusia holbrooki 64 0 0 VIII Nahuelbuta -37.882251 -73.371236 Cyprinus carpio 3 0 0 Cheirodon galusdae 31 0 0 Galaxia maculatus 70 0 0 X Puyehue -40.723249 -72.433281 Oncorhynchus mykiss 60 0 0 XIV Valdivia -39.872720 -73.160637 Gambusia holbrooki 60 0 0 TOTAL 526 0 POSITIVE SAMPLES WERE INCLUDED IN LIBRARIES FOR WGS Next generation PARTIAL sequencing using Illumina SEQUENCES MiSeq System RAW SEQUENCES ALIGNMENT (Bowtie 2) ALIGNMENT AGAINST 22 QUALITY PUBLISHED RV 531bp CONTIG ASSESSMENT SEQUENCES AND TRIMMING (Bowtie 2) PHYLOGENETIC ANALYSIS CONFIRMED THE VIRUS TO BE FV3 100% SIMILARITY SUMMARY 1 positive X. laevis (IV Region) 1 positive X. laevis (V Region) 7 positive X. laevis (Metropolitan Region) FV3 CONCLUSIONS • Ranavirus DNA in 9 X. laevis from 3 different regions. No virus in any of the native amphibians. • No Ranavirus DNA in freshwater fish, but their role as reservoirs depend on the Ranavirus type and the host fish species (Jancovich et al. 2011). • Ranavirus prevalence in Chilean amphibians and fish is lower than 5% (95% CI). • FV3 appears to be restricted to central Chile, which coincides with the African clawed-frog distribution. • Results suggest FV3 is not endemic to Chile, and that X. laevis may act as Ranavirus reservoir for native amphibians (Mora et al. 2019). • Monitoring programs at X. laevis invaded ponds, and efforts should be made to prevent the spread of this species to new water bodies. .
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