Dataset Information

TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

ABSTRACT: BACKGROUND:Avian pox is a viral disease documented in a wide range of bird species. Disease-related detrimental effects can cause dyspnea and dysphagia, and birds with high metabolic requirements, such as hummingbirds, are thus especially vulnerable to the pathogen. Hummingbirds have a strong presence in California, especially in urban environments. However, little is understood regarding the impact of pox virus on hummingbird populations. Currently, diagnosing a pox infection relies on obtaining a tissue biopsy, which poses significant risks to birds and challenges in the field. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. Our aim was to address whether pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds. METHODOLOGY AND PRINCIPAL FINDINGS:Pox-like lesion tissue, pectoral muscle, feathers, toenail clippings, blood, and swabs (both pox-like lesion tissue and non pox-like lesion tissue) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types obtained from 27 individuals. These results were compared to those of conventional PCR and comparisons were also made among sample types, utilizing lesion tissue samples as the gold standard. CONCLUSIONS AND SIGNIFICANCE:Hummingbird avian pox can be diagnosed without relying on tissue biopsies. We identify that feather samples, of which contour feathers yielded the best results, can be used for diagnosing infected birds, thus reducing sampling risk. In sum, the real-time PCR assay detected viral DNA in various integumentary system sample types and will be useful in future studies of hummingbird disease ecology.

SUBMITTER: Baek HE

PROVIDER: S-EPMC7289624 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

Baek Hanna E HE Bandivadekar Ruta R RR Pandit Pranav P Mah Michelle M Sehgal Ravinder N M RNM Tell Lisa A LA

PloS one 20200611 6

<h4>Background</h4>Avian pox is a viral disease documented in a wide range of bird species. Disease-related detrimental effects can cause dyspnea and dysphagia, and birds with high metabolic requirements, such as hummingbirds, are thus especially vulnerable to the pathogen. Hummingbirds have a strong presence in California, especially in urban environments. However, little is understood regarding the impact of pox virus on hummingbird populations. Currently, diagnosing a pox infection relies on ...[more]

PMID: 32526768

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Project description:Avian pox is a common avian virus that in its cutaneous form can cause characteristic lesions on a bird's dermal surfaces. Detection of avian pox in free-ranging birds historically relied on observations of visual lesions and/or histopathology, both which can underestimate avian pox prevalence. We compared traditional visual observation methods for avian pox with molecular methods that utilize minimally invasive samples (blood, toenail clipping, feathers, and dermal swabs) in an ecologically important group of birds, hummingbirds. Specifically, avian pox prevalence in several species of hummingbirds were examined across multiple locations using three different methods: (1) visual inspection of hummingbirds for pox-like lesions from a long-term banding data set, (2) qPCR assay of samples from hummingbird carcasses from wildlife rehabilitation centers, and (3) qPCR assay of samples from live-caught hummingbirds. A stark difference in prevalences among these three methods was identified, with an avian pox prevalence of 1.5% from banding data, 20.4% from hummingbird carcasses, and 32.5% from live-caught hummingbirds in California. This difference in detection rates underlines the necessity of a molecular method to survey for avian pox, and this study establishes one such method that could be applied to other wild bird species. Across all three methods, Anna's hummingbirds harbored significantly higher avian pox prevalence than other species examined, as did males compared with females and birds caught in Southern California compared with Northern California. After hatch-year hummingbirds also harbored higher avian pox prevalences than hatch-year hummingbirds in the California banding data set and the carcass data set. This is the first study to estimate the prevalence of avian pox in hummingbirds and address the ecology of this hummingbird-specific strain of avian pox virus, providing vital information to inform future studies on this charismatic and ecologically important group of birds.

Project description:BackgroundGene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.ResultsRNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), beta-actin (ACTB), beta-2-microglobulin (B2M), beta-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.ConclusionThe reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.

Dataset Information

TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

Publications

TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds.

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