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Bacteriological and Histopathological Findings in Cetaceans That Stranded

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Bacteriological and Histopathological Findings in Cetaceans That Stranded bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Bacteriological and histopathological findings in cetaceans 2 that stranded in the Philippines from 2017 to 2018 3 4 Marie Christine M. Obusan1, Jamaica Ann A. Caras1,2, Lara Sabrina L. Lumang1, Erika Joyce S. 5 Calderon1, Ren Mark D. Villanueva1, Cristina C. Salibay3, Maria Auxilia T. Siringan4, Windell L. 6 Rivera5, Joseph S. Masangkay6, and Lemnuel V. Aragones2 7 8 Microbial Ecology of Terrestrial and Aquatic Systems, Institute of Biology, College of Science, 9 University of the Philippines Diliman, Quezon City 1101, Philippines1 10 Marine Mammal Research Stranding Laboratory, Institute of Environmental Science and 11 Meteorology, College of Science, University of the Philippines Diliman, Quezon City 1101, 12 Philippines2 13 College of Science and Computer Studies, De La Salle University-Dasmariñas, City of Dasmariñas 14 Cavite 4115, Philippines3 15 Microbiological Research and Services Laboratory, Natural Sciences Research Institute, University of 16 the Philippines Diliman, Quezon City 1101, Philippines4 17 Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of 18 Science, University of the Philippines Diliman, Quezon City 1101, Philippines5 19 College of Veterinary Medicine, University of the Philippines Los Baños, College, Batong Malake, 20 Los Baños, Laguna 4031, Philippines6 21 22 23 Abstract 24 The relatively high frequency of marine mammal stranding events in the Philippines provide 25 many research opportunities. A select set of stranders (n=21) from 2017 to 2018 were sampled for 26 bacteriology and histopathology. Pertinent tissues and bacteria were collected from eight species (i.e. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 27 Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis 28 hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to 29 histopathological examination and antibiotic resistance screening, respectively. Lesions that were 30 observed in tissues of 19 cetaceans include congestion, hemorrhage, edema, hemosiderosis, 31 glomerulopathy, Zenker’s necrosis, atrophy, atelectasis, and parasitic cysts. These lesions may be 32 associated with factors possibly contributing to the death, debility, and stress of the animals during 33 their strandings. On the other hand, the resistance profiles of 24 bacteria (belonging to genera 34 Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans 35 were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and 36 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates 37 ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n=24) and susceptible 38 to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n=24). The 39 resistance profiles of these bacteria can be used as basis for selecting antibiotics needed in the medical 40 management of stranded cetaceans that need to be rehabilitated. Overall, the histopathological and 41 bacteriological findings of the study demonstrate the challenges faced by cetacean species in the wild, 42 such as but not limited to, biological pollution through land-sea movement of effluents, fisheries 43 interactions, and anthropogenic activities. 44 45 Keywords: cetaceans, histopathological assessment, lesions, antibiotic resistant bacteria, stranding 46 events 47 48 Introduction 49 The surveillance of wildlife health is part of an early warning system for detecting the 50 emergence or resurgence of disease threats. In the case of cetacean populations in the Philippines, 51 perhaps the most practical way of investigating their health is through their stranding events. A 52 marine mammal is considered stranded when it runs aground, or in a helpless position such as when it 53 is ill, weak, or simply lost [1]. While the event itself deserves attention, as it is not normal for any 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 54 marine mammal to strand for no apparent reason, each stranded individual can give information on 55 the abundance, distribution, health, and other ecological characteristics of its free-living counterparts 56 [2], as well as threats faced by its population [3]. It is important that stranding events be responded as 57 quickly as possible, since some stranded animals may quickly die depending on the size of the animal 58 and extent of human intervention [4]. 59 60 Biases exist in investigating the factors involved in cetacean strandings; easy-to-detect 61 circumstances such as obvious injuries (especially those intentionally inflicted by humans) are likely 62 to be more reported, whereas the role of diseases or parasites may be underestimated. The capacity to 63 detect the presence of pathogens or parasites of stranded cetaceans depends on resources, such as the 64 presence of a stranding network with the capability to respond to stranding events as well as 65 availability of expertise for conducting necropsy and other protocols for case investigation. 66 Nonetheless, whether or not a pathological condition is the underlying cause of a stranding, stranded 67 animals are good representatives for monitoring wildlife health. Also, while live stranders provide 68 good biological samples for laboratory analyses, a dead or decomposing carcass on the beach is just as 69 useful in providing specimens and other information. 70 71 The available literature on bacteria that were isolated from marine mammals worldwide 72 support the significance of investigating Gram-negative species and their antibiotic resistance or 73 susceptibility. Antibiotic susceptibility patterns have been described for populations and individuals 74 of Atlantic bottlenose dolphins, Pacific bottlenose dolphins, Risso’s dolphins, California sea lions, 75 beluga whale, sea otters and pinnipeds [5-8]. Strains of zoonotic bacteria resistant to multiple 76 antibiotics used for human and animal treatments were isolated from these animals, and some of those 77 bacteria were recognized by the American Biological Safety Association (ABSA) as human 78 pathogens. Associations between increased prevalence of antibiotic resistant bacteria in marine 79 mammals and proximity to human activities were strongly suggested [5,7,9-11]. The antibiotic 80 susceptibility profiles of bacteria isolated from cetaceans found in the Philippines where previously 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 81 reported, wherein more than half of the bacteria (n=14) had single or multiple resistances to a 82 selection of antibiotics [12]. 83 84 On the other hand, histopathological assessments proved to be useful in determining probable 85 causes of death or debility of stranded cetaceans worldwide [13-17]. Tissue lesions help confirm 86 parasitic and bacterial infections, co-morbidities, physical injuries (e.g., brought about by fisheries or 87 human interactions) and bioaccumulation of chemical compounds (e.g., persistent organic pollutants) 88 in cetaceans [16, 18-22]. Histopathological assessment is a practical and informative tool that 89 provides pathological evidence and reinforce the necropsy being done in dead cetaceans as part of the 90 stranding response. 91 92 In this study, swab and tissue samples collected from cetaceans that stranded locally from 93 February 2017-April 2018 were subjected to bacterial isolation (with subsequent antibiotic resistance 94 screening) and histopathological assessment. Data on antibiotic resistant bacteria, parasites, and tissue 95 lesions in cetaceans are valuable in evaluating the factors that may be associated with their local 96 stranding events, observed to have increased in recent years [23-24]. Out of 29 confirmed species in 97 the country, 28 species were reported to have stranded from 2005-2018 [24]. A yearly average of 105 98 cetaceans strandings occurred in the country from 2014 to 2018 [24]; 229 events were recorded by the 99 Philippine Marine Mammal Stranding Network (PMMSN) in collaboration with the Bureau of 100 Fisheries and Aquatic Resources (BFAR) from 2017 (n=121) to 2018 (n=108) involving 118 dead and 101 108 live (n=3 unknown) stranders. 102 103 Materials and methods 104 All biological samples were collected in coordination
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