Real-time reverse transcription polymerase chain reaction assay to detect a broad range of feline calicivirus isolates

In this study, a possible role of the cat flea (Ctenocephalides felis) in transmitting feline calicivirus (FCV) was examined. Fleas were fed via artificial membranes with FCV-spiked bovine blood, free of anti-FCV antibodies. Flea feces were collected daily for 10 days and incubated at room temperature. Infectivity of the feces was tested in vitro using Crandell-Reese Feline Kidney (CRFK) cells. FCV remained infectious for 8 days. These flea feces were also used to oronasally inoculate four specific pathogen-free (SPF) kittens. All kittens were successfully infected as demonstrated by virus isolation from pharyngeal swabs and seroconversion. Two of the cats showed, in addition, clinical signs. Besides the infection of cats with flea feces containing FCV, four SPF kittens were exposed to fleas that were fed with FCV-spiked bovine blood. One of the kittens was successfully infected via this route as demonstrated by virus isolation from pharyngeal swabs and virus isolation. The results of this study show that fleas can spread infectious virus through their feces or by stitch and must be considered a source of infection for uninfected cats.

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“…for a CPE. The specificity of the CPE was confirmed by a specific FCV PCR (Wilhelm and Truyen 2006).…”

Section: Virus Titration Of Fleas and Flea Fecesmentioning

confidence: 84%

Transmission of feline calicivirus via the cat flea (Ctenocephalides felis)

et al. 2009

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“…The relative abundances of feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), FIVA, and FIVC were measured with established RT-PCR protocols (10, 19, 20). PLVA proviral DNA was measured using the following RT-PCR mixture: 12.5 μl SsoFast EvaGreen SuperMix, 1 μl 8083F (10 μM) (5′-GCAGCCCTGACGGTATCC-3′), 1 μl 8165R (10 μM) (5′-GCAGTCTCCTCTGAACAATCC-3′), 5.5 μl water, and 5 μl (100 to 500 ng) template.…”

Section: Methodsmentioning

confidence: 99%

Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species

et al. 2017

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Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.

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“…Nucleic acid extraction was performed using QIAamp ® Viral RNA Mini kit (Qiagen GmbH, Hilden, Germany). Real‐time polymerase chain reaction was performed to screen for infections with FHV‐1, 20 FCV, 21 C. felis , 22 and Mycoplasma spp. 23 as per standard protocols.…”

Section: Methodsmentioning

confidence: 99%