University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2015 Prevalence Of Haemoparasites And West Nile Virus Seropositivity In Song Birds (passeriformes) From Northwest Mn And Behavioral Aspects Of Microfilariae Chad Allan Stromlund Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Stromlund, Chad Allan, "Prevalence Of Haemoparasites And West Nile Virus Seropositivity In Song Birds (passeriformes) From Northwest Mn And Behavioral Aspects Of Microfilariae" (2015). Theses and Dissertations. 1840. https://commons.und.edu/theses/1840 This Thesis is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. PREVALENCE OF HAEMOPARASITES AND WEST NILE VIRUS SEROPOSITIVITY IN SONG BIRDS (PASSERIFORMES) FROM NORTHWEST MN AND BEHAVIORAL ASPECTS OF MICROFILARIAE by Chad A. Stromlund Bachelor of Science, North Dakota State University, 2013 A Thesis Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Master of Science Grand Forks, North Dakota August 2015 1 PERMISSION Title Prevalence of Haemoparasites and West Nile Virus Seropositivity in Song Birds (Passeriformes) from Northwest MN and Behavioral Aspects of Microfilariae Department Biology Degree Master of Science In presenting this thesis in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my thesis work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this thesis or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my thesis. Chad A. Stromlund July 8, 2015 iii TABLE OF CONTENTS LIST OF FIGURES.......................................................................................................................vii LIST OF TABLES.......................................................................................................................viii ACKNOWLEDGMENTS .............................................................................................................ix ABSTRACT.....................................................................................................................................x CHAPTER I. INTRODUCTION …….....……………………………………………………….....….1 Microfilarial Enhancement………….……………………………….……………2 Sporozoite Enhancement………………………………………………………….4 West Nile Virus………………...………………………………………………….6 Mosquitoes……………….………………………………………………………..7 Songbird Hosts of West Nile Virus…….…………………………………………8 Microfilaria………………………………………………………………………..9 Plasmodium………………………………………………………………………10 Haemoproteus…………………………………………………………………....11 Leucocytozoon……………………………………………………………………12 Borellia burgdorferi………………………………………...……………………12 Trypanosomes……………………………………………………………………13 Specific Aims….…………………………………………………………………14 iv II. PREVALENCE OF HAEMOPARASITES AND WEST NILE VIRUS SEROPOSITIVITY IN SONG BIRDS (PASSERIFORMES) FROM NORTHWEST MN.......................................................................................................15 Introduction………………………………………………………………………15 Objective…………………………………………………………………………16 Methods…………………………………………………………………………..17 Site Description…………………………………………………………..17 Bird Sampling……………………………………………………………17 Bird Screening…...………………………………………………………17 Molecular Diagnosis…………………….……………………………….18 Phylogenetic Analysis….……………….………………………………..20 Results……………………………………………………………………………21 Discussion………………………………………………………………………..27 III. COMPARISON OF METHODS FOR QUANTIFYING MICROFILARIAE AND PREVALENCE OF MIDGUT PENETRATION…………………………......31 Introduction………………………………………………………………………31 Objective…………………………………………………………………...…….32 Methods…………………………………………………………………………..33 Microfilaremia of Venous Blood……………………………………...…33 Obtaining Accurate Counts of MF in Blood……………..………………33 Comparing MF Densities, Venous Blood vs Capillary Blood….………..34 Prevalence of Midgut Penetration………………………………………..35 Results……………………………………………………………………...…….36 Discussion………………………………………………………………………..37 v IV. BEHAVIORAL ASPECTS OF MICROFILARIAE………………….......................39 Introduction………………………………………………………………...…….39 Objective……………...………………………………………………………….40 Methods……….………………………………………………………………….40 Nocturnal Periodicity of Robin MF…………………………………...…40 Ability of Microfilariae to Penetrate the Midgut of Culex pipiens Mosquitoes………………………………………………………40 Luzindole Study…….……………………………………………………41 Ramelteon Study…………………………………………………………42 Results……………………………………………………………………………43 Discussion………………………………………………………………………..47 REFERENCES……………………………………………………………………………..……49 vi LIST OF FIGURES Figure Page 1. Illustration of the Invasive Process of Microfilaria in the Mosquito Midgut..............................4 2. Time Series of Mean Abundance per Breeding Bird Survey Route Adjusted for Missing Observations and Observer Variance…………………………………........................6 3. Generalized Life Cycle of a Filarial Nematode (Modified from CDC 2013)…………………10 4. Phylogenetic Tree of Haemosporidians and the Host They Infect……………………………25 5. Phylogentic Tree of Trypanosomes and the Hosts They Infect…….…………………………26 6. Line Chart Showing Nocturnal Periodicity of MF Found in the Circulatory System of American Robins……………….......……………………...………………………43 vii LIST OF TABLES Table Page 1. Primers Used to Amplify Hemoparasite DNA in Bird Blood, Roseau Co. MN, 2014.............19 2. Prevalence of Vector-Borne Hemoparasites and Seropositivity to West Nile Virus in Birds Collected in Roseau County, MN April – June, 2014……………...….......................22 3. Polyparasitism in Species with Greatest Sample Sizes Collected in Roseau County, MN April – June, 2014....................................................................................24 4. Results from Luzindole study using mosquitoes to quantify MF densities…...........................44 5. Results from Luzindole study using wing bleeds to quantify MF densities...…...…...……….44 6. Results from Ramelteon 1 study using mosquitoes to quantify MF densities……….……..…45 7. Results from Ramelteon 1 study using wing bleeds to quantify MF densities……..…………45 8. Results from Ramelteon 2 study using mosquitoes to quantify MF densities……………………46 9. Results from Ramelteon 2 study using wing bleeds to quantify MF densities..........................46 viii ACKNOWLEDGMENTS I wish to express my sincere appreciation to the members of my advisory committee for their guidance and support during my time in the master’s program at the University of North Dakota. I would especially like to thank my advisor, Jefferson Vaughan. Without him, none of this would have been possible. ix ABSTRACT Mosquitoes transmit arboviruses, haemosporidian protozoa (e.g., malaria), and filarioid nematodes. Many of these mosquito-borne pathogens occur together and transmission cycles are occurring concurrently. Despite this, mosquito pathogen transmission cycles are largely studied independently of one another. Considering the large temporal and spatial overlap among these various transmission cycles, the question becomes, what kinds of interactions are occurring between parasite transmission cycles and arboviral transmission cycles? Also, some species of mosquito are incapable of transmitting some species of arbovirus. Therefore, there must be natural barriers to prevent arbovirus infection and transmission. These barriers include the midgut barrier and the salivary gland barrier. Parasites that develop within mosquitoes have the potential to disrupt (overcome, etc.) these barriers. To further understand these interactions, I surveyed the song birds in northwest MN and screened them for haemoparasites and West Nile virus seropositivity. I focused on the behavioral aspects of microfilariae (MF) because they are known to disrupt the midgut barrier of their arthropod hosts. I found high prevalence and polyparasitism of haemoparasites infecting song birds in northwest MN. It was determined that MF of the American robin, Culex pipiens mosquitoes and West Nile virus was not an important system for enhanced viral transmission. Melatonin may play a role in the regulation of nocturnal periodicity behavior of MF, but other factors most likely contribute as well. x CHAPTER I INTRODUCTION Mosquitoes transmit arboviruses, haemosporidian protozoa (e.g., malaria), and filarioid nematodes. Many of these mosquito-borne pathogens occur together and transmission cycles are occurring concurrently. Despite this, mosquito pathogen transmission cycles are largely studied independently of one another. Considering the large temporal and spatial overlap among these various transmission cycles, the question becomes, what kinds of interactions are occurring between parasite transmission cycles and arboviral transmission cycles? To understand potential interactions, it is helpful to