Flinders Technology Associates Research Articles

Stability and Detection Limit of Avian Influenza, Newcastle Disease Virus, and African Horse Sickness Virus on Flinders Technology Associates Card by Conventional Polymerase Chain Reaction.

The Flinders Technology Associates (FTA) card, a cotton-based cellulose membrane impregnated with a chaotropic agent, effectively inactivates infectious microorganisms, lyses cellular material, and fixes nucleic acid. The aim of this study is to assess the stability and detection limit of various RNA viruses, especially the avian influenza virus (AIV), Newcastle disease virus (NDV), and African horse sickness virus (AHSV), on the FTA card, which could significantly impact virus storage and transport practices. To achieve this, each virus dilution was inoculated onto an FTA card and stored at room temperature in plastic bags for durations ranging from 1 week to 6 months. Following storage, the target genome was detected using conventional reverse transcription polymerase chain reaction. The present study demonstrated that the detection limit of AIV ranged from 1.17 to 6.17 EID50 values over durations ranging from 1 week to 5 months, while for NDV, it ranged from 2.83 to 5.83 ELD50 over the same duration. Additionally, the detection limit of AHSV was determined as 4.01 PFU for both 1 and 2 weeks, respectively. Based on the demonstrated effectiveness, stability, and safety implications observed in the study, FTA cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens.

A size and taxonomic assessment of non-lethal DNA sampling of gastropods using Flinders Technology Associates (FTA) cards

Sampling the DNA of rare animal species should have minimal impacts on individual health. This can be accomplished through non-lethal/non-invasive sampling. Few of these methods have been developed for invertebrates, including the Mollusca, which are in global decline. Tissue clipping the foot is a common non-lethal method for gastropods. However, it causes permanent damage and is inappropriate for smaller snails. This study used Flinders Technology Associates (FTA) cards to sample DNA from snail mucus for species of different sizes and habitat types, and across evolutionarily distant lineages. In a survival assay, the death rate of individuals sampled with FTA cards (12.1%) was greater than in the controls (3.7%), but the difference was not significant. Of 224 individuals representing 27 snail species (17 Hawaiian native, ten non-native) sampled using both FTA cards and tissue clipping, 80.4% of FTA samples and 91.6% of tissue samples amplified for COI, a significant difference. COI sequencing success did not differ significantly between the two methods. For individuals that failed to produce a COI sequence, an attempt was made to sequence 16S. For 16S, amplification and sequencing rates did not differ significantly between FTA and tissue samples. Habitat type and shell size did not affect FTA sampling success. Phylogenetically basal taxa exhibited lower success rates, but this may have been because of difficulty in sampling operculate taxa, and not because of identity. These results indicate that the FTA sampling is a viable non-lethal alternative to tissue clipping and can be used for diverse gastropods.

Preservation of Field-Collected Mosquito Blood Meals.

All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at -20°C or -80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.

Extracting DNA from Preserved Mosquito Blood Meals.

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.

Application of RapidHIT™ ID for cell authentication by fast and convenient STR profiling.

As cell therapies are injected directly into the body, cell authentication is essential. Short tandem repeat (STR) profiling is used for human identification in forensics as well as for cell authentication. The standard methodology (DNA extraction, quantification, polymerase chain reaction, and capillary electrophoresis) takes at least 6h and requires several instruments to obtain an STR profile. RapidHIT™ ID is a single automated instrument that provides an STR profile in 90 min. In this study, we aimed to propose a method to use RapidHIT™ ID for cell authentication. Four types of cells which are used for cell therapy or in the production process were used. The sensitivity of STR profiling was compared by the cell type and cell count using RapidHIT™ ID. Moreover, the effect of preservation solutions, pre-treatment with cell lysis solution, proteinase K, Flinders Technology Associates (FTA) cards, and dried or wet cotton swabs (with a single cell type or a mixture of two) were examined. The results were compared to those obtained by the standard methodology using genetic analyzer ThermoFisher SeqStudio. We accomplished a high sensitivity through our proposed method that can benefit cytology laboratories. Although the pre-treatment process affected the quality of the STR profile, other variables did not significantly affect STR profiling. As a result of the experiment, RapidHIT™ ID can be used as a faster and simpler instrument for cell authentication.

Visual and Rapid Detection of Escherichia coli O157:H7 in Stool Samples by FTA Card-based Loop-mediated Isothermal Amplification

Objective: Escherichia coli O157:H7 (E. coli O157:H7) can induce severe diseases in animals and humans that result in significant public health problems. Therefore, the development of rapid and visual detection methods to diagnose E. coli O157:H7 infections and monitor its prevalence is critical for the prevention and control purposes. Methods: A colorimetric loop-mediated isothermal amplification (LAMP) assay was utilized to detect E. coli O157:H7. A DNA extraction kit and Flinders Technology Associates (FTA) cards were used to extract nucleic acid in conjunction with colorimetric LAMP detection. Results: The method developed effectively distinguished E. coli O157:H7 from other pathogens with a detection limit of 25 CFU/mL in spiked stool samples. In addition, the nucleic acid of these samples was easily extracted and transported with an FTA card at room temperature. The entire detection process was completed within 35 min using simple constant-temperature equipment. Conclusion: The colorimetric LAMP method with FTA card-based nucleic acid purification was shown to rapidly detect E. coli O157:H7 with sensitivity and specificity. This visual method is expected to be widely used to control E. coli O157:H7 infections, particularly in resource-limited settings.

Sensitivity of RNA viral nucleic acid-based detection of avian influenza virus, Newcastle disease virus, and African horse sickness virus on flinders technology associates card using conventional reverse-transcription polymerase chain reaction.

The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at -80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at -80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

FTA Cards as a Rapid Tool for Collection and Transport of Infective Samples: Experience with Foot-and-Mouth Disease Virus in Libya.

Foot-and-mouth disease (FMD) is a viral disease, widespread and highly contagious, that mainly affects cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to the reduction in animal production such as a drop in milk production, loss of body weight, and a high mortality rate in young ruminants. Sixteen samples were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Flinders Technology Associates (FTA) cards impressed with blood, swabs, or vesicular epithelium samples were shipped to the WOAH FMD reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards, and molecular testing based on real-time RT-PCR assays was carried out, of which one was specifically designed for the detection of the FMD virus of serotype O, topotype O/East Africa-3 (O/EA-3), that was further confirmed by a sequence analysis of the VP1 gene. The phylogenetic analysis of the VP1 gene showed a nucleotide identity of more than 99% between the virus circulating in Libya and the FMD virus strains isolated in Algeria in 2019.

Comparison of Biosafety and Diagnostic Utility of Biosample Collection Cards.

Six different biosample collection cards, often collectively referred to as FTA (Flinders Technology Associates) cards, were compared for their ability to inactivate viruses and stabilize viral nucleic acid for molecular testing. The cards were tested with bluetongue virus, foot-and-mouth disease virus (FMDV), small ruminant morbillivirus (peste des petits ruminants virus), and lumpy skin disease virus (LSDV), encompassing non-enveloped and enveloped representatives of viruses with double-stranded and single-stranded RNA genomes, as well as an enveloped DNA virus. The cards were loaded with virus-containing cell culture supernatant and tested after one day, one week, and one month. The inactivation of the RNA viruses was successful for the majority of the cards and filters. Most of them completely inactivated the viruses within one day or one week at the latest, but the inactivation of LSDV presented a greater challenge. Three of the six cards inactivated LSDV within one day, but the others did not achieve this even after an incubation period of 30 days. Differences between the cards were also evident in the stabilization of nucleic acid. The amount of detectable viral genome on the cards remained approximately constant for all viruses and cards over an incubation period of one month. With some cards, however, a bigger loss of detectable nucleic acid compared with a directly extracted sample was observed. Using FMDV, it was confirmed that the material applied to the cards was sufficiently conserved to allow detailed molecular characterization by sequencing. Furthermore, it was possible to successfully recover infectious FMDV by chemical transfection from some cards, confirming the preservation of full-length RNAs.

Arbovirus detection in synanthropic mosquitoes from the Brazilian Amazon and in mosquito saliva using Flinders Technology Associates cards

Although arbovirus transmission and identifying target vectors may provide a baseline for planning disease control strategies, there are many gaps in knowledge regarding these mosquitoes and viral species in urban, rural, or sylvatic habitats in the Brazilian Amazon. Our goal was to screen for dengue, chikungunya, and Zika viruses in synanthropic mosquitoes and with Flinders Technology Associates (FTA) cards using insect saliva. Mosquitoes were caught using ovitraps and aspirators in the city of Porto Velho, Rondônia, Brazil. Honey-baited FTA cards were placed in mosquito cages for 15 days; whole mosquitoes and FTA cards were analysed for viral RNA using RT-qPCR assays. One pool of Aedes aegypti females was found to be infected with the Zika virus and one male mosquito was infected with dengue-4, suggesting natural vertical/venereal transmission. Our study also reported evidence of vertical/venereal transmission of ZIKV in Culex quinquefasciatus males for the first time in the Brazilian Amazon, and the feasibility of using FTA cards to detect arboviruses in the saliva of field-collected mosquitoes. Vertical/venereal transmission of viruses by atypical mosquito species reinforces the need for combined viral and entomological screening in arbovirus surveillance programs.

903 Protein Saver cards: the best alternative for dried blood spot storage at room temperature for HCV RNA

Hepatitis C virus (HCV) infection remains a global health problem, detected only in the early stages by molecular tests. Molecular tests detect HCV RNA, which is very prone to degradation by ribonucleases, reason why blood samples must be transported and stored at − 20 °C, or even − 70 °C for long-term storage. Flinders Technology Associates (FTA) cards are a useful sampling collecting device for dry blood spot (DBS) storage, especially for low and middle-income countries (LMIC). In this study, we analyzed viral HCV RNA integrity for long-term storage at room temperature compared to − 20 °C using two different types of cards for DBS: FTA Classic and 903 Protein Saver cards. For this purpose, DBS were prepared on these cards using blood or plasma samples from HCV infected patients, and samples were analysed by conventional RT-PCR. Our results showed that 903 Protein Saver cards are the best and cheapest alternative for DBS storage at room temperature. In these conditions, we found that HCV RNA integrity lasted for up to 9 months.

Putative roles of mosquitoes (Culicidae) and biting midges (Culicoides spp.) as mechanical or biological vectors of lumpy skin disease virus.

The stable fly Stomoxys calcitrans (Diptera: Muscidae) is considered as the main mechanical vector of the lumpy skin disease virus (LSDV). In addition, the mosquito species Aedes aegypti (Diptera: Culicidae) was shown to transmit the virus from donor to receptor animals. Retention of the virus for several days was shown for two additional tropical mosquito species and the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). In the present study, viral retention for 10‐ or 7‐days post feeding on virus‐spiked blood through a membrane was shown for field‐collected Aedes japonicus and laboratory‐reared Culex pipiens, two widely distributed mosquito species in temperate regions. Viral DNA could be detected from honey‐coated Flinders Technology Associates (FTA) cards and shedded faeces for 1 or 4 days after an infectious blood meal was given to Ae. aegypti. Virus increase over time and virus dissemination was observed in laboratory‐reared C. nubeculosus, but the virus could be isolated from field‐collected biting midges only from the day of exposure to the blood meal. Thus, mosquitoes might serve as mechanical vectors of LSDV in case of interrupted feeding. A putative biological virus transmission by Culicoides biting midges, as suspected from field observations, deserves further investigations.

Rapid assessment of West Nile virus circulation in a German zoo based on honey-baited FTA cards in combination with box gravid traps

BackgroundFor over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV.MethodsIn the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR.ResultsA total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus.ConclusionThe suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory.Graphical

Comparison between Whatman FTA Elute Cards and Conventional Swab for the Detection of Pathogenic Enteric Bacteria Using an RT-qPCR Assay.

The emergence of outbreaks of foodborne illness is closely associated with food contamination caused by various enteric pathogens, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus. The control of enteric pathogens poses a challenge due to the fact that these pathogens can persist for a long period of time in the environment. The rapid detection of pathogenic organisms plays a crucial role in the prevention and identification of crises related to health, safety, and well-being. Improper sample handling and processing may influence the diagnostic efficacy and accuracy. The aim of the present study was to compare the preservation capacity for enteric bacteria between Whatman Flinders Technology Associates (FTA) cards and swabs for reverse transcription-quantitative PCR (RT-qPCR) detection. It was found that Whatman FTA cards exhibited an improved preservation capacity for five types (both laboratory and environmental strains) of enteric bacteria, including Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus for RT-qPCR detection. Hence, Whatman FTA cards may be a suitable tool for the routine isolation of foodborne bacteria for molecular diagnosis. Therefore, the use of Whatman FTA cards for sample collection and preservation may increase sensitivity and accuracy for bacteria isolation and diagnosis.

Comparison of sample types from white-tailed deer (Odocoileus virginianus) for DNA extraction and analyses

Collection of biological samples for DNA is necessary in a variety of disciplines including disease epidemiology, landscape genetics, and forensics. Quantity and quality of DNA varies depending on the method of collection or media available for collection (e.g., blood, tissue, fecal). Blood is the most common sample collected in vials or on Whatman Flinders Technology Associates (FTA) cards with short- and long-term storage providing adequate DNA for study objectives. The focus of this study was to determine if biological samples stored on Whatman FTA Elute cards were a reasonable alternative to traditional DNA sample collection, storage, and extraction. Tissue, nasal swabs, and ocular fluid were collected from white-tailed deer (Odocoileus virginianus). Tissue samples and nasal swabs acted as a control to compare extraction and DNA suitability for microsatellite analysis for nasal swabs and ocular fluid extracted from FTA Elute cards. We determined that FTA Elute cards improved the extraction time and storage of samples and that nasal swabs and ocular fluid containing pigmented fluid were reasonable alternatives to traditional tissue DNA extractions.

An integrated E-Tube cap for sample preparation, isothermal amplification and label-free electrochemical detection of DNA

A simple, disposable, and integrated electronic-tube cap (E-tube cap) for DNA detection at the point-of-care was designed, fabricated, and tested. The E-tube cap contains a 3D printed electrode substrate for DNA extraction and label-free pH sensing detection. One Flinders Technology Associates (Whatman FTA) membrane was incorporated into the 3D printed electrode substrate for the isolation, concentration, and purification of DNA. The E-tube cap with captured DNA by the membrane was inserted directly into a reaction tube for loop-mediated isothermal amplification (LAMP). The isothermal amplification process was monitored in real-time by a 3D printed electrochemical electrode coated with pH-sensitive material (carbon/iridium oxide layer). The pH sensing electrode showed an excellent linear response within the pH range of 6–9 with a slope of −31.32 ± 0.5 mV/pH at room temperature. The utility of the integrated E-tube cap was demonstrated by detecting the presence of lambda DNA spiked in saliva samples with a sensitivity of 100 copies per mL sample within 30 min. Such a simple, rapid, and affordable diagnostic device is particularly suitable for point-of-care molecular diagnostics of infectious diseases.

DNA Recovery Using Ethanol-Based Liquid Medium from FTA Card-Stored Samples for HPV Detection

Introduction: Alternative methods of dry storage and transportation may be a viable alternative to the use of liquid storage medium for cervical samples, especially for screening programs in places with few resources. Objective: The objective of this study is to verify the viability and efficacy of human papillomavirus DNA (HPV-DNA) detection in cervical cell samples collected and stored on a Flinders Technology Associates (FTA) card (Whatman Indicating FTA^® Elute Micro Card) and subsequently recovered in ethanol-based liquid medium and to compare the results to those obtained using samples stored directly in ethanol-based liquid medium. Study Design: Thirty-four women submitted to ETZ (excision of the transformation zone of the cervix) were included in this study. Before ETZ, 2 samples of exfoliated cervical cells were collected from each woman by a doctor and stored in ethanol-based liquid medium and on an FTA card. DNA recovery from FTA samples was performed using ethanol-based liquid medium. Detection of HPV-DNA in the samples was performed using the Cobas® 4800 HPV Test Platform. Results and Conclusions: The HPV-DNA detection positivity rates were 70.6% for the samples collected directly in liquid medium and 64.7% for the samples stored on the FTA card, with high detection accuracy in the DNA samples recovered from the FTA card (area under the curve = 0.958; 95% confidence interval = 0.890–1.000). The concordance between the results obtained using the 2 storage media was 94.1% (Kappa = 0.866). These preliminary results suggest that collection of cervical material on an FTA card may be an alternative to storage in liquid medium since the liquid medium has some limitations. In addition, DNA recovery from the card using ethanol-based liquid medium streamlines the workflow in the laboratory and reduces the cost associated with reagents, thereby facilitating access to the HPV test in places with few resources and potentially improving cervical cancer screening.

A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples

PurposeWhatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results.MethodsTwenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography–mass spectrometry methods.ResultsWe were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent.ConclusionsThis study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood.

Comparison of three filter paper-based devices for safety and stability of viral sample collection in poultry

ABSTRACT General diagnosis of poultry viruses primarily relies on detection of viruses in samples, but many farms are located in remote areas requiring logistic transportation. Filter paper cards are a useful technology that offer an alternative for collecting and preserving samples without hazardous exposure. The goal of this study was to compare three filter papers: the Flinders Technology Associates filter (FTA®) card, dried blood spot (DBS) card and qualitative filter paper (FP) grade 2 to collect poultry samples. In particular, we have used Newcastle disease virus (NDV) to evaluate safety and a Marek’s disease virus (MDV) attenuated vaccine (CVI988) to evaluate stability of viral DNA. This experiment was divided into two parts. The first part was to determine the DNA stability and detection limit of CVI988 in samples collected in different paper supports after four storage times (3, 7, 14 and 30 days post spot). The second part was to determine the safety of papers by evaluating the viral inactivation efficacy using NDV as a representative virus. Results showed that all papers could preserve CVI988 DNA at all times, with a detection limit of 0.5 PFU/5 µl for FTA® and DBS cards, and 5 PFU/5 µl for FP. Our results showed that the NDV remained viable and infectious on the DBS card and FP, while no viable virus was detected on the FTA® card, suggesting that the FTA® card was safest to use. Therefore, the use of the DBS card and FP for infectious sample collection should be discouraged and reconsidered. RESEARCH HIGHLIGHTS The detection limits of the FTA® card, DBS card and FP for CVI988 detection were 0.5, 0.5 and 5 PFU/5 µl, respectively. All three filter papers could preserve viral DNA for at least 30 days of post spot. The DBS card and FP are not suitable for collecting NDV samples, which is one of the major economical threats for the poultry industry worldwide.

Whatman® FTA® Cards Performance for Ornithobacterium rhinotracheale DNA Amplification.

The avian pathogen Ornithobacterium rhinotracheale (ORT) has been implied in the etiology of poultry respiratory disease in recent years. To evaluate whether Whatman® Flinders Technology Associates (FTA®) cards can be used for hazard-free transport and storage of ORT samples for posterior DNA amplification, a controlled assay was performed. Three 10-fold dilutions of an ORT culture suspension were spotted on FTA cards and stored at room temperature (RT) for 6 mo. Sterile swabs were immersed in the same three 10-fold culture dilutions and stored at RT and 4 and -20 C without storage medium for the same time. DNA was extracted from both the FTA cards and swabs 1 day, 1 and 6 wk, and 6 mo following sample preparation and stored at -20 C. At the end of the experiment, real-time PCR amplification of the 16S ribosomal RNA gene was performed from DNA extracted throughout a 6-mo period from all ORT samples stored on both FTA cards and swabs. The obtained threshold cycle values for each ORT DNA extraction date were within the same range for all samples in a dilution-dependent fashion, regardless of storage temperature or used material. Pure ORT colonies could be reisolated 1 day after sample preparation from the swab dilutions stored at all temperatures but not from the FTA cards. We conclude that the efficiency of ORT DNA amplification from samples stored on FTA cards or in swabs is similar. However, FTA cards have the advantage of preventing microorganism growth, thus allowing safe transport and storage, for at least 6 mo, for bacterial dilutions down to at least 104-105 colony-forming units/ml.