Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

Margaret E. M. Farias et al...Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Methodology/Principal Findings Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study. Conclusions/Significance We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.Funding for this study was provided by: U.S. Geological Survey, Pacific Island Ecosystems Research Center (biology.usgs.gov/pierc/); U.S. Geological Survey Wildlife (biology.usgs.gov/wter/) and Invasive Species (biology.usgs.gov/invasive/) Programs; National Science Foundation (DEB0083944, www.nsf.gov); NIH/NCRR IDeA Networks of Biomedical Research Excellence (INBRE), P20RR016467 (http://www.ncrr.nih.gov/research_infrast​ructure/institutional_development_award/​idea_networks_of_biomedical_research_exc​ellence/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Real-time amplification of a serially diluted known positive sample.

<p>PCR base line subtracted curve fit data shows amplification of the <i>Avipoxvirus</i> 4b core protein gene from a 1∶2 serial dilution of first reaction PCR product using gDNA from <i>Avipoxvirus</i> culture lysate as template. The threshold for this reaction was 79.0 rfu.</p

Ct values, final intensities and sequencing results from triplicate nested TaqMan Real-Time PCR reactions for packed cell samples from wild `amakihi with presumptive pox lesions.

<p>Successful amplifications (Ct<25 and final RFU>425) are indicated in <b>bold</b>. All values are based on PCR Base Line Subtracted Curve Fit Data as calculated using iCycler version 3.1 software (BioRad). The threshold intensity was 37.0 rfu for the first reaction, 48.3 rfu for the second reaction, and 10.8 rfu for the third reaction.</p>1<p>A dash (−) indicates a failed first reaction and potential false negative, N/A indicates that no Ct value was assigned because the signal for the sample never reached the threshold intensity for that reaction.</p>2<p>Variant numbers correspond to <i>Avipoxvirus</i> clusters 1 and 2 as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010745#pone.0010745-Jarvi1" target="_blank">[8]</a>; NS indicates not sequenced.</p

Ct values and final intensities from triplicate nested TaqMan Real-Time PCR reactions for packed cell samples from wild honeycreepers with confirmed <i>Avipoxvirus</i> infections.

<p>Successful amplifications (Ct<25 and final RFU>425) are indicated in <b>bold</b>. All values are based on PCR Base Line Subtracted Curve Fit Data as calculated using iCycler version 3.1 software (BioRad). The threshold intensity was 23.4 rfu for the first reaction, 51.4 rfu for the second reaction, and 41.7 rfu for the third reaction.</p>1<p>Samples are identified by species, island and year of capture. Abbreviations are as follows: HAAM, Hawaìi `amakihi (<i>Hemignathus virens</i>); IIWI, ìiwi (<i>Vestiaria coccinea</i>); APAP, `apapane (<i>Himatione sanguinea</i>); HI, Hawaìi; MO, Molokài.</p>2<p>Laboratory <i>Avipoxvirus</i> isolates cultured from these individuals are included in Jarvi, et al., 2008 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010745#pone.0010745-Jarvi1" target="_blank">[8]</a>.</p>3<p>NR indicates the sample was not included in that reaction, a dash (−) indicates a failed first reaction and potential false negative, N/A indicates that no Ct value was assigned because the signal for the sample never reached the threshold intensity for that reaction.</p

Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay.

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats

Summary of the rat identification and measurements recorded during the trial including gender, number of worms recovered at necropsy, body weights prior to infection and at 6 weeks PI, spleen measurements and treatment group (C control, LD low dose, HD high dose).

<p>*The mean number of adult worms isolated from HD rats was significantly greater (<i>p</i> = 0.001, F = 10.11, DF = 2) than those isolated from LD or C groups. The mean spleen weight of HD rats was significantly greater (<i>p</i> = 0.011, F = 5.92. DF = 2) than uninfected controls.</p><p>** Rats in trial I were ~5 months of age and rats in trial II were ~6 months of age at the time of body weight measurement.</p><p>Summary of the rat identification and measurements recorded during the trial including gender, number of worms recovered at necropsy, body weights prior to infection and at 6 weeks PI, spleen measurements and treatment group (C control, LD low dose, HD high dose).</p

Results of qPCR for detection of RLW DNA in blood or tissue of rats involved in Trial I and Trial II.

<p>High dose infection (HD, 50 larvae/ml gavage), low dose (LD, 10 larvae/ml gavage), control (C, 0 larvae/ml gavage).</p><p>¹One extraction per individual except wk 6 tail: tail tissue, which had two extractions per individual for controls, 3 extractions per individual for infected.</p><p>²Positive = clear signal in large proportion of replicates</p><p>³If the number of replicate qPCR tests differed between the two individuals, they are reported separately.</p><p>Results of qPCR for detection of RLW DNA in blood or tissue of rats involved in Trial I and Trial II.</p

Mean number of adult worms isolated from heart and lungs.

<p>The mean number of adult worms isolated from the heart and lungs of HD rats was significantly higher (<i>p</i> = 0.001, F = 10.11, DF = 2) than that isolated from LD or control groups. Worm numbers ranged from 0–16 in the LD group and 8–57 in the HD group. Mean worm numbers of control rats was 0, of LD rats was 5.9, and of HD rats was 26. Standard error bars are indicated.</p

Mean spleen weights (g) of control, low dose and high dose rats.

<p>Mean spleen weight of the HD was significantly greater (<i>p</i> = 0.011, F = 5.92. DF = 2) than the uninfected controls. Mean spleen weight of control rats was 202.8 mg, of LD rats was 332.9 mg and of HD rats was 373.3 mg. Standard error bars are indicated.</p