Dataset Information

Detection and isolation of exotic Newcastle disease virus from field-collected flies.

ABSTRACT: Flies were collected by sweep net from the vicinity of two small groups of "backyard" poultry (10-20 chickens per group) that had been identified as infected with exotic Newcastle disease virus (family Paramyxoviridae, genus avulavirus, ENDV) in Los Angeles County, CA, during the 2002-2003 END outbreak. Collected flies were subdivided into pools and homogenized in brain-heart infusion broth with antibiotics. The separated supernatant was tested for the presence of ENDV by inoculation into embryonated chicken eggs. Exotic Newcastle disease virus was isolated from pools of Phaenicia cuprina (Wiedemann), Fannia canicularis (L.), and Musca domestica L., and it was identified by hemagglutination inhibition with Newcastle disease virus antiserum. Viral concentration in positive pools was low (<1 egg infectious dose50 per fly). Isolated virus demonstrated identical monoclonal antibody binding profiles as well as 99% sequence homology in the 635-bp fusion gene sequence compared with ENDV recovered from infected commercial egg layer poultry during the 2002 outbreak.

SUBMITTER: Chakrabarti S

PROVIDER: S-EPMC7107500 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Detection and isolation of exotic Newcastle disease virus from field-collected flies.

Chakrabarti Seemanti S King Daniel J DJ Afonso Claudio C Swayne David D Cardona Carol J CJ Kuney Douglas R DR Gerry Alec C AC

Journal of medical entomology 20070901 5

Flies were collected by sweep net from the vicinity of two small groups of "backyard" poultry (10-20 chickens per group) that had been identified as infected with exotic Newcastle disease virus (family Paramyxoviridae, genus avulavirus, ENDV) in Los Angeles County, CA, during the 2002-2003 END outbreak. Collected flies were subdivided into pools and homogenized in brain-heart infusion broth with antibiotics. The separated supernatant was tested for the presence of ENDV by inoculation into embryo ...[more]

PMID: 17915517

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Project description:BackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.MethodsField-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.ResultsThe qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (?Cq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionsThis study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

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Detection and isolation of exotic Newcastle disease virus from field-collected flies.

Publications

Detection and isolation of exotic Newcastle disease virus from field-collected flies.

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