Fish Farmageddon: the Infectious Salmon Aquacalypse
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A Guide to Culturing Parasites, Establishing Infections and Assessing Immune Responses in the Three-Spined Stickleback
ARTICLE IN PRESS Hook, Line and Infection: A Guide to Culturing Parasites, Establishing Infections and Assessing Immune Responses in the Three-Spined Stickleback Alexander Stewart*, Joseph Jacksonx, Iain Barber{, Christophe Eizaguirrejj, Rachel Paterson*, Pieter van West#, Chris Williams** and Joanne Cable*,1 *Cardiff University, Cardiff, United Kingdom x University of Salford, Salford, United Kingdom { University of Leicester, Leicester, United Kingdom jj Queen Mary University of London, London, United Kingdom #Institute of Medical Sciences, Aberdeen, United Kingdom **National Fisheries Service, Cambridgeshire, United Kingdom 1Corresponding author: E-mail: [email protected] Contents 1. Introduction 3 2. Stickleback Husbandry 7 2.1 Ethics 7 2.2 Collection 7 2.3 Maintenance 9 2.4 Breeding sticklebacks in vivo and in vitro 10 2.5 Hatchery 15 3. Common Stickleback Parasite Cultures 16 3.1 Argulus foliaceus 17 3.1.1 Introduction 17 3.1.2 Source, culture and infection 18 3.1.3 Immunology 22 3.2 Camallanus lacustris 22 3.2.1 Introduction 22 3.2.2 Source, culture and infection 23 3.2.3 Immunology 25 3.3 Diplostomum Species 26 3.3.1 Introduction 26 3.3.2 Source, culture and infection 27 3.3.3 Immunology 28 Advances in Parasitology, Volume 98 ISSN 0065-308X © 2017 Elsevier Ltd. http://dx.doi.org/10.1016/bs.apar.2017.07.001 All rights reserved. 1 j ARTICLE IN PRESS 2 Alexander Stewart et al. 3.4 Glugea anomala 30 3.4.1 Introduction 30 3.4.2 Source, culture and infection 30 3.4.3 Immunology 31 3.5 Gyrodactylus Species 31 3.5.1 Introduction 31 3.5.2 Source, culture and infection 32 3.5.3 Immunology 34 3.6 Saprolegnia parasitica 35 3.6.1 Introduction 35 3.6.2 Source, culture and infection 36 3.6.3 Immunology 37 3.7 Schistocephalus solidus 38 3.7.1 Introduction 38 3.7.2 Source, culture and infection 39 3.7.3 Immunology 43 4. -
The 2014 Golden Gate National Parks Bioblitz - Data Management and the Event Species List Achieving a Quality Dataset from a Large Scale Event
National Park Service U.S. Department of the Interior Natural Resource Stewardship and Science The 2014 Golden Gate National Parks BioBlitz - Data Management and the Event Species List Achieving a Quality Dataset from a Large Scale Event Natural Resource Report NPS/GOGA/NRR—2016/1147 ON THIS PAGE Photograph of BioBlitz participants conducting data entry into iNaturalist. Photograph courtesy of the National Park Service. ON THE COVER Photograph of BioBlitz participants collecting aquatic species data in the Presidio of San Francisco. Photograph courtesy of National Park Service. The 2014 Golden Gate National Parks BioBlitz - Data Management and the Event Species List Achieving a Quality Dataset from a Large Scale Event Natural Resource Report NPS/GOGA/NRR—2016/1147 Elizabeth Edson1, Michelle O’Herron1, Alison Forrestel2, Daniel George3 1Golden Gate Parks Conservancy Building 201 Fort Mason San Francisco, CA 94129 2National Park Service. Golden Gate National Recreation Area Fort Cronkhite, Bldg. 1061 Sausalito, CA 94965 3National Park Service. San Francisco Bay Area Network Inventory & Monitoring Program Manager Fort Cronkhite, Bldg. 1063 Sausalito, CA 94965 March 2016 U.S. Department of the Interior National Park Service Natural Resource Stewardship and Science Fort Collins, Colorado The National Park Service, Natural Resource Stewardship and Science office in Fort Collins, Colorado, publishes a range of reports that address natural resource topics. These reports are of interest and applicability to a broad audience in the National Park Service and others in natural resource management, including scientists, conservation and environmental constituencies, and the public. The Natural Resource Report Series is used to disseminate comprehensive information and analysis about natural resources and related topics concerning lands managed by the National Park Service. -
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DISEASES OF AQUATIC ORGANISMS Published July 30 Dis Aquat Org Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. 11: Gyrodactylus sp. J. L. Tojo*, M. T. Santamarina Department of Microbiology and Parasitology, Laboratory of Parasitology, Faculty of Pharmacy, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain ABSTRACT: A total of 24 drugs were evaluated as regards their efficacy for oral treatment of gyro- dactylosis in rainbow trout Oncorhj~nchusmykiss. In preliminary trials, all drugs were supplied to infected fish at 40 g per kg of feed for 10 d. Twenty-two of the drugs tested (aminosidine, amprolium, benznidazole, b~thionol,chloroquine, diethylcarbamazine, flubendazole, levamisole, mebendazole, n~etronidazole,mclosamide, nitroxynil, oxibendazole, parbendazole, piperazine, praziquantel, roni- dazole, secnidazole, tetramisole, thiophanate, toltrazuril and trichlorfon) were ineffective Triclabenda- zole and nitroscanate completely eliminated the infection. Triclabendazole was effective only at the screening dosage (40 g per kg of feed for 10 d), while nitroscanate was effective at dosages as low as 0.6 g per kg of feed for 1 d. KEY WORDS: Gyrodactylosis . Rainbow trout Treatment. Drugs INTRODUCTION to the hooks of the opisthohaptor or to ulceration as a result of feeding by the parasite. The latter is the most The monogenean genus Gyrodactylus is widespread, serious. though some individual species have a restricted distri- Transmission takes place largely as a result of direct bution. Gyrodactyloses affect numerous freshwater contact between live fishes, though other pathways species including salmonids, cyprinids and ornamen- (contact between a live fish and a dead fish, or with tal fishes, as well as marine fishes including gadids, free-living parasites present in the substrate, or with pleuronectids and gobiids. -
Fish and Fishery Products Hazards and Controls Guidance Fourth Edition – APRIL 2011
SGR 129 Fish and Fishery Products Hazards and Controls Guidance Fourth Edition – APRIL 2011 DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION CENTER FOR FOOD SAFETY AND APPLIED NUTRITION OFFICE OF FOOD SAFETY Fish and Fishery Products Hazards and Controls Guidance Fourth Edition – April 2011 Additional copies may be purchased from: Florida Sea Grant IFAS - Extension Bookstore University of Florida P.O. Box 110011 Gainesville, FL 32611-0011 (800) 226-1764 Or www.ifasbooks.com Or you may download a copy from: http://www.fda.gov/FoodGuidances You may submit electronic or written comments regarding this guidance at any time. Submit electronic comments to http://www.regulations. gov. Submit written comments to the Division of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852. All comments should be identified with the docket number listed in the notice of availability that publishes in the Federal Register. U.S. Department of Health and Human Services Food and Drug Administration Center for Food Safety and Applied Nutrition (240) 402-2300 April 2011 Table of Contents: Fish and Fishery Products Hazards and Controls Guidance • Guidance for the Industry: Fish and Fishery Products Hazards and Controls Guidance ................................ 1 • CHAPTER 1: General Information .......................................................................................................19 • CHAPTER 2: Conducting a Hazard Analysis and Developing a HACCP Plan -
Viral Haemorrhagic Septicaemia Virus (VHSV): on the Search for Determinants Important for Virulence in Rainbow Trout Oncorhynchus Mykiss
Downloaded from orbit.dtu.dk on: Nov 08, 2017 Viral haemorrhagic septicaemia virus (VHSV): on the search for determinants important for virulence in rainbow trout oncorhynchus mykiss Olesen, Niels Jørgen; Skall, H. F.; Kurita, J.; Mori, K.; Ito, T. Published in: 17th International Conference on Diseases of Fish And Shellfish Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Olesen, N. J., Skall, H. F., Kurita, J., Mori, K., & Ito, T. (2015). Viral haemorrhagic septicaemia virus (VHSV): on the search for determinants important for virulence in rainbow trout oncorhynchus mykiss. In 17th International Conference on Diseases of Fish And Shellfish: Abstract book (pp. 147-147). [O-139] Las Palmas: European Association of Fish Pathologists. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. DISCLAIMER: The organizer takes no responsibility for any of the content stated in the abstracts. -
Fish Welfare on Scotland's Salmon Farms
FISH WELFARE ON SCOTLAND’S SALMON FARMS A REPORT BY ONEKIND Lorem ipsum CONTENTS 1 INTRODUCTION 2 6.3.1 Increased aggression 26 6.3.2 Increased spread of disease and parasites 26 2 SALMON SENTIENCE 6.3.2 Reduced water quality 26 AND INDIVIDUALITY 4 6.3.4 Issues with low stocking densities 27 2.1 Fish sentience 5 6.4 Husbandry 27 2.2 Atlantic salmon as individuals 5 6.4.1 Handling 27 6.4.2 Crowding 28 3 ATLANTIC SALMON LIFE CYCLE 6 6.4.3 Vaccination 28 3.1 Life cycle of wild salmon 6 6.5 Transportation 28 3.2 Life cycle of farmed Alantic Salmon 6 6.6 Failed smolts 29 6.7 Housing 20 4 SALMON FARMING IN SCOTLAND 8 6.8 Slaughter 31 5 KEY WELFARE ISSUES 10 7 MARINE WILDLIFE WELFARE IMPACTS 32 5.1 High mortality rates 10 7.1 Wild salmon and trout 32 5.2 Sea lice 11 7.2 Fish caught for salmon food 33 5.2.1 How do sea lice compromise 7.3 Seals 33 the welfare of farmed salmon? 11 7.4 Cetaceans 34 5.2.2 How are sea lice levels monitored? 12 7.5 Crustaceans 34 5.2.3 How severe is sea lice infestation in Scotland? 13 8 FUTURE CHALLENGES 35 5.3 Disease 14 8.1 Closed containment 35 5.3.1 Amoebic Gill Disease 15 8.2 Moving sites offshore 35 5.3.2 Cardiomyopathy Syndrome 15 5.3.3 Infectious salmon anaemia 15 9 ACCREDITATION SCHEMES 5.3.4 Pancreas disease 15 AND STANDARDS 36 5.4 Treatment for sea lice and disease 16 9.1 Certification in Scotland 36 5.4.1 Thermolicer 17 9.2 What protection do standards provide salmon? 36 5.4.2 Hydrolicer 17 9.2.1 Soil Association Organic standards 36 5.4.3 Hydrogen peroxide 17 9.2.2 RSPCA Assured 36 5.5 Cleaner fish 18 -
Predatory Impact of Aurelia Aurita on the Zooplankton Community in The
Barz and Hirche: Predatory impact of Aurelia aurita on the zooplankton community in the Bornholm Basin (central Baltic) CM 2004/J: 12 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Not to be cited without prior reference to the authors CM 2004/J:12 Predatory impact of Aurelia aurita on the zooplankton community in the Bornholm Basin (central Baltic) Kristina Barz and Hans-Jürgen Hirche Alfred-Wegener-Institute for Polar and Marine Research Columbusstrasse, 27568 Bremerhaven, Germany tel.: +49 471 4831 1324, fax: +49 471 4831 1918 [email protected] Abstract The annual cycle of abundance and vertical distribution of the scyphozoan medusa Aurelia aurita was studied in the Bornholm Basin (central Baltic) in 2002. Seasonal changes in prey composition and predatory impact were investigated by analysing stomach contents. The dominant prey organisms of A. aurita were several cladoceran species (Bosmina coregoni maritima, Evadne nordmanni and Podon spp.). They represented up to 93% of the food organisms. Bivalve larvae and copepods (Centropages hamatus, Temora longicornis, Acartia spp., Pseudocalanus acuspes, Eurytemora hirundoides and Oithona similis) were less frequently found. Nauplii and copepodites I-III were not eaten by the medusae neither were fish eggs and larvae used as prey. Based on average medusae and zooplankton abundance, the predatory impact was very low. In August, when mean abundance of A. aurita was highest, only 0.1% of the copepod and 0.54% of the cladoceran standing stock were eaten per day. However in regions -
Salmon Aquaculture Dialogue Working Group Report on Salmon Disease
Salmon Aquaculture Dialogue Working Group Report on Salmon Disease Larry Hammell - Atlantic Veterinary College, University of Prince Edward Island, Canada Craig Stephen- Centre for Coastal Health, University of Calgary, Canada Ian Bricknell- School of Marine Sciences, University of Maine, USA Øystein Evensen- Norwegian School of Veterinary Medicine, Oslo, Norway Patricio Bustos- ADL Diagnostic Chile Ltda., Chile With Contributions by: Ricardo Enriquez- University of Austral, Chile 1 Citation: Hammell, L., Stephen, C., Bricknell, I., Evensen Ø., and P. Bustos. 2009 “Salmon Aquaculture Dialogue Working Group Report on Salmon Disease” commissioned by the Salmon Aquaculture Dialogue, available at http://wwf.worldwildlife.org/site/PageNavigator/SalmonSOIForm Corresponding author: Larry Hammell, email: [email protected] This report was commissioned by the Salmon Aquaculture Dialogue. The Salmon Dialogue is a multi-stakeholder, multi-national group which was initiated by the World Wildlife Fund in 2004. Participants include salmon producers and other members of the market chain, NGOs, researchers, retailers, and government officials from major salmon producing and consuming countries. The goal of the Dialogue is to credibly develop and support the implementation of measurable, performance-based standards that minimize or eliminate the key negative environmental and social impacts of salmon farming, while permitting the industry to remain economically viable The Salmon Aquaculture Dialogue focuses their research and standard development on seven key areas of impact of salmon production including: social; feed; disease; salmon escapes; chemical inputs; benthic impacts and siting; and, nutrient loading and carrying capacity. Funding for this report and other Salmon Aquaculture Dialogue supported work is provided by the members of the Dialogue‘s steering committee and their donors. -
Mote Marine Laboratory Red Tide Studies
MOTE MARINE LABORATORY RED TIDE STUDIES FINAL REPORT FL DEP Contract MR 042 July 11, 1994 - June 30, 1995 Submitted To: Dr. Karen Steidinger Florida Marine Research Institute FL DEPARTMENT OF ENVIRONMENTAL PROTECTION 100 Eighth Street South East St. Petersburg, FL 33701-3093 Submitted By: Dr. Richard H. Pierce Director of Research MOTE MARINE LABORATORY 1600 Thompson Parkway Sarasota, FL 34236 Mote Marine Laboratory Technical Report No. 429 June 20, 1995 This document is printed on recycled paper Suggested reference Pierce RH. 1995. Mote Marine Red Tide Studies July 11, 1994 - June 30, 1995. Florida Department of Environmental Pro- tection. Contract no MR 042. Mote Marine Lab- oratory Technical Report no 429. 64 p. Available from: Mote Marine Laboratory Library. TABLE OF CONTENTS I. SUMMARY. 1 II. CULTURE MAINTENANCE AND GROWTH STUDIES . 1 Ill. ECOLOGICAL INTERACTION STUDIES . 2 A. Brevetoxin Ingestion in Black Seabass B. Evaluation of Food Carriers C. First Long Term (14 Day) Clam Exposure With Depuration (2/6/95) D. Second Long Term (14 Day) Clam Exposure (3/21/95) IV. RED TIDE FIELD STUDIES . 24 A. 1994 Red Tide Bloom (9/16/94 - 1/4/95) B. Red Tide Bloom (4/13/94 - 6/16/95) C. Red Tide Pigment D. Bacteriological Studies E. Brevetoxin Analysis in Marine Organisms Exposed to Sublethal Levels of the 1994 Natural Red Tide Bloom V. REFERENCES . 61 Tables Table 1. Monthly Combined Production and Use of Laboratory C. breve Culture. ....... 2 Table 2. Brevetoxin Concentration in Brevetoxin Spiked Shrimp and in Black Seabass Muscle Tissue and Digestive Tract Following Ingestion of the Shrimp ............... -
Working Group on Pathology and Diseases of Marine Organisms (WGPDMO)
ICES WGPDMO REPORT 2018 AQUACULTURE STEERING GROUP ICES CM 2018/ASG:01 REF. ACOM, SCICOM Report of the Working Group on Pathology and Diseases of Marine Organisms (WGPDMO) 13-17 February 2018 Riga, Latvia International Council for the Exploration of the Sea Conseil International pour l’Exploration de la Mer H.C. Andersens Boulevard 44–46 DK-1553 Copenhagen V Denmark Telephone (+45) 33 38 67 00 Telefax (+45) 33 93 42 15 www.ices.dk [email protected] Recommended format for purposes of citation: ICES. 2018. Report of the Working Group on Pathology and Diseases of Marine Or- ganisms (WGPDMO), 13-17 February 2018, Riga, Latvia. ICES CM 2018/ASG:01. 42 pp. https://doi.org/10.17895/ices.pub.8134 The material in this report may be reused using the recommended citation. ICES may only grant usage rights of information, data, images, graphs, etc. of which it has own- ership. For other third-party material cited in this report, you must contact the origi- nal copyright holder for permission. For citation of datasets or use of data to be included in other databases, please refer to the latest ICES data policy on the ICES website. All extracts must be acknowledged. For other reproduction requests please contact the General Secretary. The document is a report of an Expert Group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council. © 2018 International Council for the Exploration of the Sea ICES WGPDMO REPORT 2018 | i Contents Executive summary ............................................................................................................... -
Proteome Analysis Reveals a Role of Rainbow Trout Lymphoid Organs During Yersinia Ruckeri Infection Process
www.nature.com/scientificreports Correction: Author Correction OPEN Proteome analysis reveals a role of rainbow trout lymphoid organs during Yersinia ruckeri infection Received: 14 February 2018 Accepted: 30 August 2018 process Published online: 18 September 2018 Gokhlesh Kumar 1, Karin Hummel2, Katharina Noebauer2, Timothy J. Welch3, Ebrahim Razzazi-Fazeli2 & Mansour El-Matbouli1 Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. Head kidney and spleen are major lymphoid organs of the teleost fsh where antigen presentation and immune defense against microbes take place. We investigated proteome alteration in head kidney and spleen of the rainbow trout following Y. ruckeri strains infection. Organs were analyzed after 3, 9 and 28 days post exposure with a shotgun proteomic approach. GO annotation and protein-protein interaction were predicted using bioinformatic tools. Thirty four proteins from head kidney and 85 proteins from spleen were found to be diferentially expressed in rainbow trout during the Y. ruckeri infection process. These included lysosomal, antioxidant, metalloproteinase, cytoskeleton, tetraspanin, cathepsin B and c-type lectin receptor proteins. The fndings of this study regarding the immune response at the protein level ofer new insight into the systemic response to Y. ruckeri infection in rainbow trout. This proteomic data facilitate a better understanding of host-pathogen interactions and response of fsh against Y. ruckeri biotype 1 and 2 strains. Protein-protein interaction analysis predicts carbon metabolism, ribosome and phagosome pathways in spleen of infected fsh, which might be useful in understanding biological processes and further studies in the direction of pathways. Enteric redmouth disease (ERM) causes signifcant economic losses in salmonids worldwide. -
Detection of Paramoeba Perurans in Scotish Marine Wild Fish Populations
Bull. Eur. Ass. Fish Pathol., 35(6) 2015, 217 NOTE ȱȱParamoeba perurans in Ĵȱȱ ȱęȱ H. E. B. Stagg*, M. Hall, I. S. Wallace, C. C. Pert, S. Garcia Perez and C. Collins Marine Scotland Science, Marine Laboratory, Aberdeen, AB11 9DB Abstract ȱȱParamoeba perurans, ȱȱȱȱȱȱ ȱȱ¢ȱȱ ȱ ȱęȱȱĴȱȱ ȱǻȱƽȱŘǰřŚŞǼǯȱOverall, the apparent prevalence was low. A ȱęǰȱȱȱȱTrachurus trachurus, ȱǯȱȱȱȱęȱȱȱȱ ȱP. perurans in horse mackerel. Paramoeba perurans is an amoeba parasite and the Salmo salar and rainbow trout Oncorhynchus ȱȱȱȱȱȱǻ Ǽȱ mykiss (Munday et al., 1990); coho salmon O. (Young et al., 2007, Crosbie et al., 2012). The kisutchȱǻ ȱȱǯǰȱŗşŞŞǼDzȱ Scophthalmus ȱ ȱęȱȱȱȱȱŘŖŖŜȱ maximus ǻ¢ȱȱǯǰȱŗşşŞǼDzȱȱȱDicen- with additional outbreaks occurring since 2011 trarchus labrax (Dykova et al., 2000); chinook ȱȱȱ¢ȱ ȱȱȱęȱ salmon O. tshawytscha ǻȱȱǯǰȱŘŖŖŞǼDzȱ ȱȱȱĴȱȱ¢ȱ ayu Plecoglossus altivelis (Crosbie et al., 2010); (Marine Scotland Science unpublished data). ballan wrasse Labrus bergylta (Karlsbakk et al., ȱȱȱȱęȱȱȱ 2013); blue warehou Seriolella brama (Adams (Shinn et al., 2014) especially in the Australian ȱǯǰȱŘŖŖŞǼDzȱȱȱȱDiplodus puntazzo ȱȱ¢ȱȱȱ (Dykova and Novoa, 2001). ȱȱȱȱȱęȱȱȱ ŗşŞŚȱǻ¢ǰȱŗşŞŜǼǯȱȱȱȱȱȱ ȱȱȱ ȱęȱȱȱȱȱȱ reported in the USA (Kent et al., ŗşŞŞǼǰȱ ȱ P. peruransȱȱȱȱȱȱȱȱ (Rodger and McArdle, 1996), the Mediterranean ȱ¢ȱȱȱȱȱȱȱ ǻ¢ȱȱǯǰȱŗşşŞǼǰȱ ȱȱǻȱȱ ȱȱȱ ȱȱȱȱ ǯǰȱŘŖŖŞǼǰȱ ¢ȱǻȱȱǯǰȱŘŖŖŞǼǰȱ ȱ ȱȱęǯȱȱǰȱP. perurans has only (Crosbie et al., 2010), Chile (Bustos et al., 2011) ȱȱȱęȱȱȱ- ȱȱȱȱǻȱȱǯǰȱŘŖŗŚǼǯȱ- ǯȱȱȱȱȱȱ¢ȱȱ ceptible species to AGD include: Atlantic salmon ȱȱ ȱParamoeba ǯȱȱ ȱęȱ * Corresponding author’s e-mail: [email protected] ŘŗŞǰȱǯȱǯȱǯȱȱǯǰȱřśǻŜǼȱŘŖŗś ǻȱȱǯǰȱŘŖŖŞǼȱ ȱȱȱ ȱ ȱȱȱȱȱ¢ȱȱȱ ȱȱȱȱ¢ȱȱȱȱ ȱ ȱȱęȱȱȱ¢ȱǻ ȱ ȱȱ in Tasmania and tested ȱǯǰȱŘŖŖŗǼǯȱ¢ȱęȱ ȱȱ ȱ using histological and immunohistochemical each haul based on the approximate proportion techniques however, the amoeba species was ȱȱȱȱȱȱǰȱȱ ȱȱȱȱȱȱȱȱȱ ȱ ȱȱȱȱȱ ȱȱP.