Carter T Atkinson 20200424 tabular digital data Hilo Hawaii Cooperative Studies Unit Atkinson, C. T. 2020. Hawaiian Island forest bird avian malaria detection using whole blood preserved in lysis buffer, 2005-2006. U.S. Geological Survey data release, https://doi.org/10.5066/P98CVJJG. https://doi.org/10.5066/P98CVJJG Carter Atkinson 2020 publication HCSU Technical Report Series 094 Atkinson, C. T. 2020. Use of whole blood samples preserved in DNA lysis buffer for serological detection of avian malaria in Hawaiian forest birds. Technical Report 094, Hawaii Cooperative Studies Unit, University of Hawaii at Hilo PENDING It is unclear whether DNA lysis buffers used for preservation of whole blood samples from Hawaiian forest birds cause denaturation and loss of antigen binding capability of antibody molecules. If their antigen binding capability is not affected, then the samples can be used in serological assays to provide an independent assessment of the accuracy of PCR (polymerase chain reaction) diagnostic tests for avian malaria. This data set provides derived Percent ELISA (Enzyme linked Immunosorbant Assay) Values calculated from adjusted absorbance measurements that were collected with a BioRad Model 3550 ELISA plate reader at a wavelength setting of 405 nm. Recent detections of avian malarial parasites in native and non-native forest birds at Hakalau Forest National Wildlife Refuge and reports of epidemic transmission of the disease in high elevation habitats (Freed et al. 2005) as well as controversy over accuracy of the PCR diagnostic test that was being used (Jarvi et al. 2002) led to a request by U.S. Fish and Wildlife Service to see if existing blood samples that were preserved in a DNA lysis buffer could be used for independent confirmation of the findings with antibody based serological methods (Jarvi et al. 2002). The primary objective of this study was to test whether some DNA buffers used for preservation of blood samples (Feldman et al. 1995, Jarvi et al. 2002) cause denaturation and loss of antigenicity of antibody molecules. If the buffer does not destroy antigenicity of these molecules, then the samples can be used in serological assays to provide an independent assessment of the accuracy of the PCR test. REFERENCES: (1) Feldman, R. A., L. A. Freed, and R. L. Cann. 1995. A PCR test for avian malaria in Hawiian birds. Molecular Ecology 4: 663-673. (2) Freed, L. A., R. L. Cann, M. L. Goff, W. A. Kuntz, and G. R. Bodner. 2005. Increase in avian malria at upper elevation in Hawai`i. The Condor 107: 753-764. (3) Jarvi, S. I., J. J. Schultz, and C. T. Atkinson. 2002. PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines. Journal of Parasitology 88: 153-158. 20050101 20060101 observed Complete None planned -156.2640 -154.6820 20.3395 18.8023 Hawaii Island ISO 19115 Topic Category biota None immunoblot enzyme linked immunosorbant assay Hawaii Amakihi Chlorodrepanis virens polymerase chain reaction PCR ELISA Lysis Buffer Western blot avian malaria Plasmodium relictum USGS Metadata Identifier USGS:5e98e22b82ce172707f6f981 None Hakalau Forest National Wildlife Refuge Hawaii Hawaiian Islands None None Carter T Atkinson U.S. Geological Survey, NW-PACIFIC ISLAND REG Research Microbiologist mailing address

Bldg 344 Chain of Craters Rd.

Hawaii National Park HI 96718 US 808-985-6401 808-967-8568 catkinson@usgs.gov This dataset and associated technical report were supported by the U.S. Geological Survey Science Support Program and the U. S. Geological Survey Wildlife and Terrestrial Resources Program. Environment as of Metadata Creation: Microsoft Windows 10 Enterprise; Microsoft Excel Version 1902 (Build 11328.20468 Click-to-Run) None Chlorodrepanis virens Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Tetrapoda Class Aves Order Passeriformes Family Fringillidae Genus Chlorodrepanis Species Chlorodrepanis virens TSN: 997800 No formal tests were conducted to verify values collected by the BioRad Model 5500 Microplate Reader. All derived values were proofed for accuracy before any analysis. No formal logical accuracy tests were conducted Some Percent (%) ELISA values are missing because spectrophotometer readings exceeded the upper measurement limits of the spectrophotometer. Data set is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details. N/A N/A Work was conducted at the U.S. Geological Survey, Kilauea Field Station, Hawai`i Volcanoes National Park between 2005 and 2006. Blood samples were collected by jugular venipuncture from seven captive Hawai`i `Amakihi (Chlorodrepanis virens) with chronic experimental infections with the avian blood parasite, Plasmodium relictum plus one uninfected bird. Infected birds were survivors from prior experimental studies and still had detectable numbers of circulating parasites by microscopy of Giemsa-stained blood smears. The birds were held under University of Hawaii IACUC Protocol 00-035-5 for experimental studies of avian malaria and pox virus. Up to 100 µl of heparinized whole blood was drawn from each of the birds and divided into three equal portions. One part was mixed 1:10 with phosphate buffered saline, pH 7.0, (PBS) as an untreated positive control for serological analysis. The second portion was mixed 1:10 with a DNA buffer used by Feldman et al. (1995) (TEN; 10 mM Tris, 10 mM NaCl, 2 mM EDTA), pH 8.0) and frozen. The third portion was mixed 1:10 with a DNA lysis buffer used by Jarvi et al. (2002) (SDS-EDTA; 0.1 M Tris-HCl, pH 8.0, 0.1 M EDTA, 2% SDS) and frozen. REFERENCES: Feldman, R. A., L. A. Freed, and R. L. Cann. 1995. A PCR test for avian malaria in Hawiian birds. Molecular Ecology 4: 663-673. Jarvi, S. I., J. J. Schultz, and C. T. Atkinson. 2002. PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines. Journal of Parasitology 88: 153-158. 20200319 Carter T Atkinson U.S. Geological Survey, NW-PACIFIC ISLAND REG Research Microbiologist mailing address

Bldg 344 Chain of Craters Rd.

Hawaii National Park HI 96718 US 808-985-6401 808-967-8568 catkinson@usgs.gov The DNA samples were subsequently thawed several weeks after being frozen. Samples that were preserved in SDS-EDTA buffer were dialysed overnight against ice cold PBS in dialysis tubing (Sigma D-0405, molecular weight cutoff = 12,400) to attempt to remove SDS and EDTA (Dong et al. 1997). After removal from the tubing the following morning, the liquid was centrifuged 15 min at 28,200 g to pellet any cellular debris, transferred to Centricon YM-50 centrifugal filter devices with a nominal molecular weight limit of 50,000 daltons (Millipore Corporation, Billerica, MA), and centrifuged 15 min at 13,000 g to concentrate antibodies. If samples preserved in SDS-EDTA buffer were too gelatinous for use in the Centricon filters, they were briefly sonicated on wet ice to liquefy the pellet with a Microson XL200 ultrasonic cell disruptor (Medsonic, Inc., Farmingdale, NY). The concentrate was removed from the upper wells of the filters, transferred to vials, and either refrigerated or frozen prior to evaluation by ELISA (enzyme linked immunosorbent assay). Samples preserved in TEN buffer and PBS were used without additional processing. 20200319 Carter T Atkinson U.S. Geological Survey, NW-PACIFIC ISLAND REG Research Microbiologist mailing address

Bldg 344 Chain of Craters Rd.

Hawaii National Park HI 96718 US 808-985-6401 808-967-8568 catkinson@usgs.gov For the ELISA analysis, samples were serially diluted with PBS at 1/10, 1/20, 1/40, 1/80, 1/160, 1/320, 1/640, and 1/1280 and then tested by a modification of the ELISA procedure described by Graczyk et al. (1993). Briefly, a crude P. relictum antigen extract was prepared from infected Pekin duckling erythrocytes by lysing the cells with saponin to rupture erythrocyte membranes, by pelleting the lysed cells and by washing them with sodium carbonate buffer, pH 9.0, to remove hemoglobin. The cells were pelleted and sonicated to disrupt cellular membranes and centrifuged at 20,000 g to pellet cellular debris. The supernatant was used to coat individual wells of ELISA plates (Immulon 4HBX flat bottom plates, Thermo Fisher Scientific, Waltham, MA) by overnight incubation at 4°C. The ELISA procedure followed that described by Graczyk et al. (1993), using a rabbit anti-chicken IgG alkaline phosphatase conjugate (Sigma-Aldrich Corp., St. Louis, MO, catalog number A9171) that cross-reacts with native forest bird IgG as a secondary antibody. Raw absorbance values were measured with a BioRad Model 5500 Microplate Reader at a wavelength of 405 nm. Measurements were made of the different sample dilutions (three replicates per dilution), blank wells that were incubated with test reagents alone (6 replicates per plate), and wells containing positive and negative control duckling plasma (3 replicates of each per plate). REFERENCES: Graczyk, T. K., M. R. Cranfield, and C. J. Shiff. 1993. ELISA method for detecting anti-Plasmodium relictum and anti-Plasmodium elongatum antibody in infected duckling sera using Plasmodium falciparum antigens. Journal of Parasitology 79: 879-885. 20200319 Adjusted absorbance values for each replicate were calculated by subtracting the mean absorbance value of 6 blank wells (incubated with all reagents except the test plasma) from each raw absorbance value for individual ELISA plates, including positive and negative controls. 20200319 Mean Adjusted absorbance values were calculated for duplicate or triplicate adjusted values for each sample dilution and triplicate positive and triplicate negative control from respective ELISA plates. 20200319 To account for plate to variability in ELISA measurements and allow for comparisons among ELISA plates, the mean adjusted absorbance value for replicates of each sample was expressed as a percentage of positive and negative Pekin duckling plasma controls that were run on each plate. This value, the percent ELISA Value (%EV), was calculated as (mean absorbance of triplicate samples – the mean absorbance of triplicate negative controls)/(mean absorbance of triplicate positive controls – mean absorbance of triplicate negative controls)*100. 20200319 Carter T Atkinson U.S. Geological Survey, NW-PACIFIC ISLAND REG Research Microbiologist mailing address

Bldg 344 Chain of Craters Rd.

Hawaii National Park HI 96718 US 808-985-6401 808-967-8568 catkinson@usgs.gov ELISA Values.csv CSV text file Producer Defined Treatment Experimental Treatment Producer Defined PBS Sample diluted in Phosphate Buffered Saline Producer defined SDS Sample diluted with SDS (sodium dodecyl sulfate - ethylenediaminetetracetic acid) buffer Producer defined TEN Sample diluted with TEN (Tris-Ethylenediaminetetraacetic Acid) Buffer Producer defined TEN+Protk+SDS Sample diluted with TEN (Tris-Ethylenediaminetetraacetic Acid) Buffer with Proteinase K and SDS (sodium dodecyl sulfate - ethylenediaminetetracetic acid) added after freezing and thawing Producer defined Plate 96 well ELISA Plate Number Producer Defined 06-77b ELISA Plate number 06-077b Producer defined 06-77c ELISA Plate number 06-77c Producer defined 06-78a ELISA Plate number 06-78a Producer defined 06-78b ELISA Plate number 06-78b Producer defined 06-78c ELISA Plate number 06-78c Producer defined 06-119a ELISA Plate number 06-119a Producer defined 06-119b ELISA Plate number 06-119b Producer defined 06-119c ELISA Plate number 06-119c Producer defined 06-121a ELISA Plate number 06-121a Producer defined 06-121b ELISA Plate number 06-121b Producer defined 06-121c ELISA Plate number 06-121c Producer defined ELISA Plate number 15-194 ELISA Plate number 15-194 Producer defined Status Infection Status Producer Defined Y Infected with Plasmodium relictum Producer defined N Uninfected with Plasmodium relictum Producer defined Band Unique leg band number of individual Hawaii Amakihi that provided test plasma Producer Defined 130 Hawaii Amakihi Band Number 130 Producer defined 137 Hawaii Amakihi Band Number 137 Producer defined 139 Hawaii Amakihi Band Number 139 Producer defined 142 Hawaii Amakihi Band Number 142 Producer defined 147 Hawaii Amakihi Band Number 147 Producer defined 148 Hawaii Amakihi Band Number 148 Producer defined 77735 Hawaii Amakihi Band Number 77735 Producer defined 6 Hawaii Amakihi Band Number 6 Producer defined Dilution (1:10) 1 microliter of sample + 9 microliters of diluent Producer Defined . raw absorbance measurement exceeded upper limit of ELISA Plate Reader Producer defined -2.143 702.168 % ELISA Value 0.001 Dilution (1:20) 1 microliter of sample + 19 microliters of diluent Producer Defined -1.286 521.739 % ELISA Value 0.001 Dilution (1:40) 1 microliter of sample + 39 microliters of diluent Producer Defined -1.571 216.937 % ELISA Value 0.001 Dilution (1:80) 1 microliter of sample + 79 microliters of diluent Producer Defined -2.000 183.211 % ELISA Value 0.001 Dilution (1:160) 1 microliter of sample + 159 microliters of diluent Producer Defined -2.258 94.845 % ELISA Value 0.001 Dilution (1:320) 1 microliter of sample + 319 microliters of diluent Producer Defined -4.065 60.236 % ELISA Value 0.001 Dilution (1:640) 1 microliter of sample + 539 microliters of diluent Producer Defined -5.420 25.331 % ELISA Value 0.001 Dilution (1:1280) 1 microliter of sample + 1279 microliters of diluent Producer Defined -6.267 9.720 % ELISA Value 0.001 These data report derived Percent ELISA Values (%EV) that were calculated from adjusted absorbance values (ELISA Values.csv) from blood samples collected from Hawaii Amakihi that were infected with malaria or from an uninfected control bird. Atkinson, C. T. 2020. Use of whole blood samples preserved in DNA lysis buffer for serological detection of avian malaria in Hawaiian forest birds. Hawaii Cooperative Studies Unit Technical Report 094. GS ScienceBase U.S. Geological Survey mailing address

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Denver CO 80225 United States 1-888-275-8747 sciencebase@usgs.gov Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Digital Data https://doi.org/10.5066/P98CVJJG None 20200827 Carter T Atkinson U.S. Geological Survey, NW-PACIFIC ISLAND REG Research Microbiologist mailing address

Bldg 344 Chain of Craters Rd.

Hawaii National Park HI 96718 US 808-985-6401 808-967-8568 catkinson@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999