Natalia Ochoa, Erica A. Miller, Andrea Howey-Newcomb, and Maria S. Sepúlveda ABSTRACT METHODS RESULTS: DNA ISOLATION & IDENTIFICATION Passerines are commonly parasitized by helminths and apicomplexans at the

time they enter bird rehabilitation centers. Only a handful of studies have been published on the parasites of wild passerines commonly housed in rehabilitation centers in eastern states. It is important to know what species of parasites the birds are infected with since some may constitute species that could turn out to be pathogenic. The objective of this study was to conduct a survey of helminths and coccidia from in-patient passerines at Tri-State Bird Rescue & Research in Newark, Delaware. In July and August of 2018, fecal samples were collected biweekly from several pens housing a variety of passerines. Fecal flotations were conducted and eggs classified into major groups (nematode, cestode and

coccidia). Although a few cases of cestode and nematode infections were recorded, coccidia were by far the most common and were mostly detected in pens housing house finches (Haemorhous mexicanus), American robins (Turdus migratorius) and common grackles (Quiscalus quiscula). In order to determine the coccidia species, DNA was isolated from highly infected fecal samples. Primers were designed to target Eimeria species commonly found in passerines.[1] DNA was sequenced and sequences blasted against known coccidian species reported in passerines from North America.

INTRODUCTION

✺ Coccidia are the largest group of endoparasites in passerines and in most bird species. (See Figure 2 for life cycle) ✺ Since coccidia species vary greatly in pathogenicity,[2] it is important to be able to identify them ✺ Several studies have reported various Eimeria spp. from passerine and poultry populations world-wide [3] ✺

However, identifying coccidia parasites to species is not straightforward and non- invasive tools are needed for their diagnosis from live birds Objective ✺ Identify Eimeria species present in fecal samples

collected from various in-patient passerines through PCR in addition to documenting the presence of other endoparasites Hypothesis ✺ Proper PCR methods and primer design should reveal presence of Eimeria species in infected fecal samples

Figure 2. Three main stages of Eimeria reproduction – merogony, gametogony and

sporogony.[4]

Figure 1. Coccidia oocysts from passerine patients at Tri-State Bird

Rescue & Research, Inc. DNA Identification ✵ A total of 5 primers were tested corresponding to 5 Eimeria species ✵ There was no DNA amplification with any of

the selected primers 15 !m ✺ Coccidia was the most prevalent of documented endoparasites ✺

Although DNA was isolated from fecal samples, no amplification occurred ✺ Additional fecal samples need to be collected from an expanded pool of bird species ✺ Different primer sets should be developed to enhance coccidia species detection ability ✺ DNA isolation technique should be refined to increase DNA yield and quality

REFERENCES 1. Berto BP, Flausino W, McIntosh D, Teixeira WL, Lopes CWG. (2011) Coccidia

of New World passerine birds (Aves: Passeriformes): a review of Eimeria Schneider, 1875 and Isospora Schneider, 1881 (Apicomplexa: Eimeriidae). Sys Parasitol 80:159–204. 2. Knight A, Ewen JG, Brekke P, Santure AW. (2018) Chapter Two – The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds. Advances in Parasitolgy 99: 35- 60. 3. Medina JP, Miranda-Salgado C, García-Conejo M, Galindo-Sánchez KP, Mejía-García JC, Janczur KM, Gomes Lopes CW, Berto BP,

Soriano-Vargas E. (2015) Coccidia in passerines from the Nevado de Toluca

National Park, Mexico. Acta Parasitologica. 60: 173-174. 4. Price KR. (2012) Use of live

vaccines for coccidiosis control in

replacement layer pullets. The Journal of Applied Poultry Research. 21: 679-692.

Identification of Coccidian Species in Passerines from Tri

Bird Rescue & ✵ Highly infected fecal samples (1000+ oocysts per slide) were subject to DNA

extraction ✵ Sample was initially flushed with 5.75 % sodium hypochlorite (bleach) to weaken coccidia oocyst cell walls prior to DNA extraction ✵ Primers were designed to bind to unique DNA sequences present in each Eimeria species (See Table 1) ✵ Fecal samples were collected biweekly between July and August 2018 ✵ A total

of 44 samples were collected. All

were floated with Fecasol®

Figure 3.Percent prevalence of coccidia, nematode and cestode

species in fecal samples collected from July 5 to August 14, 2018.Time

range was divided into three consecutive periods (07/05-07/19,

07/23-08/04, 08/07- 08/14). Samples were collected from pens housing

passerines in both inside and outdoor settings. The number of samples

collected is shown next to the enclosure’s setting.

Research,

Inc. in Northern Delaware, USA RESULTS: PARASITE PREVALENCE

f Table 1. Five primers targeted to different Eimeria species.

30 !m30 !m 25 !m Figure 4. Coccidia oocysts from an outside enclosure

Figure 5. Capillaria (nematode) egg from an indoor enclosure Figure 6. Cestode egg from an indoor enclosure DNA Isolation ✵ DNA was extracted from highly infected fecal samples and measured with NanoDrop ND-2000 spectrometer (Thermo Scientific) (See Figure 7)

CONCLUSIONS FUTURE DIRECTIONS

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State Figure 7. DNA extraction results measured by NanoDrop

ND-2000 spectrometer.