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Universidad Nacional del Comahue – San Carlos de Bariloche, Argentina.
(Machine Translated)
1 First Mass Mortality of Marine Mammals Caused by Highly Pathogenic Influenza Virus
2 (H5N1) in South America
Víctor Gamarra-Toledo1,2, Pablo I. Plaza1*
, Giancarlo Inga3,4, Roberto Gutiérrez2,3 3 ,
Oscar García-Tello3
, Leonela Valdivia-Ramírez3
, Deyvis Huamán-Mendoza3 4 , José C.
Nieto-Navarrete3
, Sandra Ventura3
, Sergio A. Lambertucci1 5
6
7 1 Grupo de Investigaciones en Biología de la Conservación, Laboratorio Ecotono,
8 INIBIOMA, Universidad Nacional del Comahue – CONICET, Quintral 1250
9 (R8400FRF), San Carlos de Bariloche, Argentina.
10
11 2 Museo de Historia Natural (MUSA), Universidad Nacional de San Agustín de
12 Arequipa, Av. Alcides Carrión s/n, Arequipa,
13 Perú.
14
3 15 Servicio Nacional de Áreas Naturales Protegidas por el Estado (SERNANP). Calle
16 Diecisiete 355, Urb. El Palomar, San Isidro, Lima, Perú.
17
18 4 Asociación Convive Perú, Puerto Maldonado, Madre de Dios, Perú.
19
20 Corresponding author: plazapablo22@gmail.com 21 perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 22 ABSTRACT 23 We report the first worldwide infection and massive mortality associated with a Highly 24 Pathogenic Influenza Virus (H5N1) in sea lions of Peru. The transmission pathway of 25 H5N1 may have been through the close contact of sea lions with infected wild birds. 26 We cannot rule out direct transmission among sea lions. perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 27 The recent global epidemic event (2020-2022) caused by the highly pathogenic avian 28 influenza (HPAI) A (H5N1) is the largest observed so far, several global outbreaks 29 having been caused (1,2). This is the first time the presence of this pathogen has been 30 registered in some regions, where it has produced massive mortality in wild birds (2–4). 31 This is of great concern and suggests a change has taken place in the dynamics of this 32 pathogen (2). 33 At the end of 2022, the H5N1 virus reached South America (Peru, Ecuador, Colombia, 34 Venezuela and Chile), with alarming outcomes in Peru (4). This pathogen was detected 35 for the first time in wild birds in Peru on 13 November, as it caused very huge mortality 36 levels (>22,000 birds over 4 weeks within protected areas) (4). Reports on both 37 protected and non-protected areas suggest the virus generated a disease that killed at 38 least >50,000 wild birds by the end of 2022, particularly Peruvian pelicans (Pelecanus 39 thagus) and Peruvian boobies (Sula variegata) (4, Gamarra-Toledo et al. unpublished 40 data). Most birds showed signs of neurological problems (e.g., incoordination, tremors 41 and partial convulsions) before dying; impressive images of sandy beaches with dozens 42 of dying birds highlighted the magnitude of this catastrophe (Suppl Mat, 43 https://www.youtube.com/watch?v=45UZVY4IxTY). The large biomass of infected 44 wild birds may have led to a spillover event affecting predators and scavengers, 45 including marine mammals cohabiting with them, as reported in other parts of the world 46 (5). In this article, we report the death of 634 sea lions (mainly Otaria flavescens and a 47 few Arctocephalus australis) on Peruvian coasts over 5 weeks, and give details of the 48 analyses that prove their infection with the Highly Pathogenic Influenza Virus (H5N1). 49 The Study 50 During January and the first days of February 2023, more than six hundred sea lions 51 were found dead or dying on Peruvian beaches (e.g., Asia, Chorrillos, Cerro Azul and perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 52 Chilca beaches) and in protected areas (e.g., Reserva Nacional Paracas, Reserva 53 Nacional San Fernando, Reserva Nacional Sistema de Islas, Islotes y Puntas Guaneras) 54 (Fig. 1, Table 1). The high mortality observed was worrisome; for instance, up to 100 55 dead individuals floating together in the sea – an unprecedented observation for this 56 geographical region (Fig. 1, Table 1). The clinical symptoms of dying individuals were 57 mainly neurological, such as tremors, convulsions and paralysis. They also showed 58 respiratory signs such as dyspnea, tachypnea, nasal and buccal secretions and 59 pulmonary edema. Most dead animals were female, and several abortions were 60 observed. There are no other records of such high mortality of aggregated sea lions. 61 Individuals were examined by veterinarians and some dead animals were necropsied 62 (see Fig. 2 A, B). The body condition of the sea lions necropsied ranged from good to 63 very good, suggesting they died due to an acute health problem. Substantial quantities 64 of whitish secretions filling the upper respiratory tracts (trachea and pharynx) were 65 observed in the necropsies and in dying animals (Fig. 2 C), which explains the severe 66 dyspnea and tachypnea clinically observed by veterinarians. Lungs were congestive, 67 with hemorrhagic focus compatible with interstitial pneumonia. Brains were also 68 congestive with hemorrhagic focus compatible with encephalitis, which explains the 69 neurological signs observed in dying individuals (Fig. 2 D). The small intestine showed 70 necrotic focus compatible with duodenitis. Of epidemiological and conservation 71 concern, a total of 634 dead individuals belonged mainly to Otaria flavescens (n=630) 72 and some to Arctocephalus australis (n=4). However, the latter species was not tested 73 and there are no other records of high mortality in this species, so we cannot confirm the 74 actual cause of their death. At the time of writing this dispatch (February 2023) sea 75 lions mortalities continue. perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 76 Given the epidemiologic situation produced by H5N1 in wild birds that cohabit with 77 these sea lions (4), samples were taken in a subset of individuals to diagnose this virus. 78 To diagnose HPIV, samples of Otaria flavescens (n=6; including 3 samples from 79 different parts of each animal) were taken inside a protected area (Reserva Nacional 80 Paracas) and sent to the Avian Pathology Laboratory of the Universidad Nacional 81 Mayor de San Marcos. Samples consisted of organs such as kidneys, spleen and lungs, 82 stomach contents, and oral, nasal and rectal swabs (Fig 2 E). Real time RT-PCR 83 targeting the Influenza A matrix segment were performed: all the tested individuals 84 were positive for influenza A (Table 1). Through posterior RT-PCR analysis using 85 specific primers to identify the hemagglutinin and neuraminidase, this virus was 86 characterized as Influenza A of the H5N1 subtype, the virus that had produced massive 87 mortality of wild birds in previous months (4). Almost at the same time (between 88 January 18 and 26) SERFOR (Servicio Nacional Forestal y de Fauna Silvestre) took 89 another 6 samples from dead animals outside protected areas (beaches of the provinces 90 of Lima and Cañete), following the same procedure. These samples were analyzed at 91 the Genomics Laboratory of the Pontificia Universidad Católica del Perú. Three sea lion 92 samples also tested positive for HPAI H5N1 (OFICIO-0017-2023-MIDAGRI93 SENASA-DSA), and a dolphin (Tursiops truncatus) was also reported as testing 94 positive in Piura, in the northern region of Peru. 95 Conclusions 96 We show that sea lions (Otaria flavescens) of Peru, an endangered species according to 97 the national legislation (6), were infected by HPAI (H5N1) and developed a deadly 98 associated disease producing massive mortality in several regions of the Peruvian 99 coastline (Fig. 1). The sea lion mass mortality described is compatible with systemic 100 HPIV that resulted in acute encephalitis and pneumonia. Some specific cases of perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 101 infection and mortality of marine mammals due to HPIV with similar clinical and 102 anatomopathological characteristics have been reported in previous studies around the 103 world (5,7,8). For instance, 1,400 dead harbor seals (Phoca vitulina) were found dead in 104 Germany alone; necropsied individuals (n=17) showed congestive lungs and interstitial 105 pneumonia due to HPIV (7). Similarly, in USA, seal mortalities were reported with 106 pneumonia and neurological symptoms due to H5N1 (5). However, to our knowledge, 107 this is the first confident report of massive mortality by disease associated with HPIV 108 (H5N1) in sea lions, and the first report of wild mammal mortality in South America. 109 The source of the HPAI affecting these sea lions was very probably the large number of 110 infected birds/carcasses on the Peruvian coastline (4). Sea lions may be infected by 111 close contact with these carcasses and even through their consumption (see Fig. 2 F). 112 However, the transmission pathway remains unknown until now. 113 Such high levels of mortality in a social animal are worrisome. We cannot exclude 114 direct transmission among sea lions due to their colonial breeding, and because many 115 animals died simultaneously in groups. In fact, based on recent research suggesting the 116 first mammal-to-mammal infection in minks (Neovison vison) (9) and the large number 117 of sea lions currently affected, we cannot rule out that the virus has adapted to mammals 118 and that sea lion-sea lion transmission has begun in Peru; this should be urgently 119 investigated. Moreover, the mass mortality of animals that can weigh around 350 kg 120 (10) produces an enormous biomass of infected tissue, which could perpetuate the 121 transmission of H5N1 and other pathogens. This could have serious consequences for 122 the ecosystem and human health. 123 Further research is required to address the transmission pathway in social mammals. 124 This is particularly important in our case, given the complexities of the H5N1 Peruvian 125 outbreak; i.e., severe mortality of wild birds, potential wild bird spillover to mammals, perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 126 dead animals on beaches with human presence and human interaction with infected 127 animals (see Supplementary Material Video 1). Since the emergence of the 128 A/goose/Guangdong/1/1996 (Gs/Gd) H5N1 influenza A virus in China in 1996, 129 different Highly Pathogenic Influenza Viruses (HPIV) have produced thousands of 130 human infections with a lethality of 50% (3,11). We would like to call attention to the 131 fact that in this geographical region of the world, human–infected animal interaction is 132 common, so infections might begin to rise (there is already one case of a 9-year-old girl 133 in Ecuador; https://www.eldiario.com.ar/242825-se-confirmo-el-primer-caso-de-gripe134 aviar-en-nina-de-9-anos-en-ecuador/) and this must be addressed if we are to avoid the 135 risk of a pandemic through evolutionary emergence (12). 136 References 137 1. Wille M, Barr IG. Resurgence of avian influenza virus. Science. 2022 Apr 138 29;376(6592):459–60. 139 2. Harvey JA, Mullinax JM, Runge MC, Prosser DJ. The Changing Dynamics of Highly 140 Pathogenic Avian Influenza H5N1: Next Steps for Management & Science in North 141 America. 2022; 142 3. Ramey AM, Hill NJ, DeLiberto TJ, Gibbs SE, Camille Hopkins M, Lang AS, et al. 143 Highly pathogenic avian influenza is an emerging disease threat to wild birds in 144 North America. The Journal of Wildlife Management. 2022;86(2):e22171. 145 4. Gamarra-Toledo V, Plaza PI, Gutiérrez R, Luyo P, Hernani L, Angulo F, et al. Avian 146 flu threatens Neotropical birds. Science. 2023;379(6629):246–246. 147 5. Puryear W, Sawatzki K, Hill N, Foss A, Stone JJ, Doughty L, et al. Outbreak of 148 Highly Pathogenic Avian Influenza H5N1 in New England Seals. bioRxiv. 149 2022;2022–07. 150 6. Ministerio de Agricultura y Riego. Decreto Supremo N° 004-2014-MINAGRI. El 151 Peruano. 2014;pp.520497– 520504. 152 7. Bodewes R, Bestebroer TM, van der Vries E, Verhagen JH, Herfst S, Koopmans MP, 153 et al. Avian influenza A (H10N7) virus–associated mass deaths among harbor seals. 154 Emerging infectious diseases. 2015;21(4):720. 155 8. Shin DL, Siebert U, Lakemeyer J, Grilo M, Pawliczka I, Wu NH, et al. Highly 156 pathogenic avian influenza A (H5N8) virus in gray seals, Baltic Sea. Emerging 157 infectious diseases. 2019;25(12):2295. perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 158 9. Agüero M, Monne I, Sánchez A, Zecchin B, Fusaro A, Ruano MJ, et al. Highly 159 pathogenic avian influenza A (H5N1) virus infection in farmed minks, Spain, 160 October 2022. Eurosurveillance. 2023;28(3):2300001. 161 10. Vaz-Ferreira R. South American sea lion Otaria flavescens (Shaw, 1800). Handbook 162 of marine mammals. 1981;1:39–65. 163 11. Shi J, Zeng X, Cui P, Yan C, Chen H. Alarming situation of emerging H5 and H7 164 avian influenza and effective control strategies. Emerging Microbes & Infections. 165 2023;12(1):2155072. 166 12. Tripathi A, Dhakal HC, Adhikari K, Chandra Timsina R, Wahl LM. Estimating the 167 risk of pandemic avian influenza. Journal of Biological Dynamics. 2021;15(1):327– 168 41. 169 170 171 perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 172 Figure 1. Map of geographical distribution of high mortalities in sea lions in January 173 and February 2023 and sampling localities (green triangle and yellow star) for sea lions 174 testing positive of Highly Pathogenic Influenza Virus (H5N1) on the Peruvian coastline 175 (SERFOR = Servicio Nacional Forestal y de Fauna Silvestre; SERNANP = Servicio 176 Nacional de Áreas Naturales Protegidas por el Estado). 177 178 179 Figure 1. Map of geographical distribution of high mortality in sea lions and sampling localities for testing of Highly Pathogenic Influenza Virus (H5N1) in sea lions on the Peruvian coastline in January and February 2023 (SERFOR = Servicio Nacional Forestal y de Fauna Silvestre; SERNANP = Servicio Nacional de Áreas Naturales Protegidas por el Estado). 216-217 1-73 120-145 Sea lion mortality (number of individuals) Sea lion carcasses floating in the sea (n=100) Samples taken by SERFOR Samples taken by SERNANP perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 180 Figure 2. Images showing the field work and sample collection for testing of Highly 181 Pathogenic Influenza Virus (H5N1) in sea lions in the Paracas National Reserve on 182 the Peruvian coastline, February 2023. A) Sea lion carcasses on the beach; B) Dying 183 sea lion with ataxia; C) Dead sea lion with avian flu symptoms; D) Sample collection 184 for RT-PCR; E) Sea lion necropsy; F) Sea lion trapping and eating an infected 185 Guanay cormorant on January 23th of 2023 (Photo: Sandra Lizarme) in the Reserva 186 Nacional Paracas. 187 Figure 2. A C B D E F perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this 188 Table 1. Sea lion mortality and positive tests of Highly Pathogenic Influenza Virus 189 (H5N1) in protected areas of Peru between January and February 2023. Abbreviations 190 for the Natural Protected Areas are as follows: RNSF = Reserva Nacional San 191 Fernando, RNILLESCAS = Reserva Nacional Illescas, RNPARACAS = Reserva 192 Nacional Paracas, ZRANCÓN = Zona Reservada Ancón, RNSIIPG = Reserva Nacional 193 Sistema de Islas, Islotes y Puntas Guaneras. Species Date Natural Protected Area Island & Guano island Symptoms Total deaths South American Fur Seal (Arctocephalus australis) 26-Jan RNSF 3 5-Feb RNPARACAS Yuyos, Caracolita, Lagunilla, La Roja 1 South American Sea Lion (Otaria flavescens) 6-Jan RNILLESCAS 38 14-Jan RNPARACAS Lagunillas, Supay, Atenas Eyes closed and watering, nosebleeds and difficulty breathing 3 16-Jan ZRANCÓN 4 18-Jan RNPARACAS La Mina 1 19-Jan RNPARACAS Aguada 1 RNSIIPG Isla Cavinzas 1 20-Jan RNPARACAS Lagunillas, La Roja, Yumaque, Atenas 32 21-Jan RNPARACAS Santo Domingo 1 RNSIIPG Punta Lomitas Foaming at the mouth, convulsions, paralysis of the forelimbs 1 23-Jan RNPARACAS Barlovento 6 Isla San Gallan 33 RNSIIPG Isla Cavinzas 3 Isla Pachacamac 7 24-Jan RNSIIPG Isla Cavinzas 4 25-Jan RNPARACAS Lagunillas 36 RNSIIPG Isla Cavinzas 6 Isla Asia 20 26-Jan RNPARACAS Los viejos 24 perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this RNSF Carcasses floating in the sea 5 RNSIIPG Punta San Juan Convulsions (dying individuals) 2 27-Jan RNSF 20 RNSIIPG Isla Pescadores 2 Isla Asia Carcasses floating in the sea 100 Punta San Juan 8 ZRANCÓN 3 28-Jan RNPARACAS Arquillo 1 29-Jan RNSIIPG Punta San Juan 2 30-Jan RNPARACAS Ancla Dyspnea, tremors (dying individuals) 17 Caclio 4 Chucho 4 La Raya 12 Punta Prieto 3 Rancherio 5 31-Jan RNPARACAS Santo Domingo 12 Sequion Ataxia, tremors (dying individuals) 2 1-Feb RNPARACAS Yumaque 5 2-Feb RNPARACAS Karwas, La Raya Dyspnea, tremors (dying individuals) 9
3-Feb RNSF 188
4-Feb RNPARACAS Yumaque Punta
Ballena 3
5-Feb RNPARACAS
Yuyos,
Caracolita,
Lagunilla, La
Roja
2
Total 634
194 *6 individuals tested, and all results were positive for the Highly Pathogenic Influenza
195 Virus (H5N1)
perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
bioRxiv preprint doi: https://doi.org/10.1101/2023.02.08.527769; this version posted February 10, 2023. The copyright holder for this