Development of a Semi-Nested Reverse Transcription Polymerase Chain Reaction Assay for the Retrospective Diagnosis of Canine Distemper Virus Infection

Abstract

Abstract. Formalin-fixed, paraffin-embedded tissue blocks from 14 dogs were used to test the utility of a newly developed semi-nested reverse transcription polymerase chain reaction (RT-PCR) assay for canine distemper virus (CDV). The results from this new test were compared with those of histopathologic examination, fluorescent antibody detection (FA), and immunohistochemistry (IHC). The semi-nested RT-PCR protocol was used to detect CDV RNA in 9 of the 10 cases that were positive by at least 1 of the immunologic (FA and IHC) methods. Sequence data indicated that the amplified strains of CDV are more closely related to a naturally occurring strain than to a vaccine strain. Thus, the semi-nested RT-PCR assay is a useful diagnostic method applicable to the retrospective diagnosis of CDV infection. Sequence determination may yield molecular epidemiologic information regarding vaccine efficacy.Canine distemper virus (CDV) is a member of the genus Morbillivirus, family Paramyxoviridae, which are negative-sense, single-stranded RNA viruses. 3 Canine distemper virus is a highly contagious pathogen with a worldwide distribution. 3,7 Eradication of CDV is considered impossible because of the virus's wide host range, which includes terrestrial carnivores and marine mammals. 1,3,8,14,17,19 Because of its pantropic nature, neurologic, respiratory, and integumentary disorders are among the many presentations of canine distemper infection. 21 Accurate diagnostic assays for CDV are of paramount importance considering its global distribution, its broad host range, its myriad of clinical presentations, its history of mass mortality events, 3,7,8,17 and the availability of vaccines that may curtail local epizootics. 4 There are currently several techniques that can be used to test for CDV infection in dogs, including immunofluorescence, 5,12 immunohistochemistry, 11,15 and histopathologic analysis. 15,21 However, each of these tests has limitations; immunofluorescence can only be performed on fresh-frozen tissue, immunohistochemistry is dependent on specific antibody-fixation parameters, and histopathology is not specific and requires a confirmatory test. Furthermore, none of these tests allow for genetic analysis. Consequently, the use of multiple tests may be the best diagnostic strategy, and it is important to develop new diagnostic assays that allow retrospective genetic analysis and maintain or improve detection limits. Modern infectious disease diagnostic innovations include the development of nucleic acid-based assays, 6,9,16,18 which amplify pathogen-specific nucleic acid, permitting the detection of fewer organisms and thereby increasing the probability of detecting the agent. Previous studies have shown that CDV RNA can be reverse transcribed and then amplified from whole blood, serum, cerebrospinal fluid, and formalinfixed, paraffin-embedded (FFPE) tissues. 6,18 The action of formalin results in chemical modifications to RNA that tend to limit the size of transcribable nucleic acid fragments. 10 To compensate for the ...

Retrospective Differentiation of Canine Distemper Virus and Phocine Distemper Virus in Phocids

“…Tissues included lung (harbor, harp, and hooded seals), kidney (harbor and harp seals), brain (harp seal), pancreas (Caspian seal), spleen (Caspian seal), stomach (harp seal), liver (harp seal), skin (harp seal), and esophagus (harp seal). Positive controls were tissues from two dogs infected with canine distemper virus (Stanton et al, 2002). Tissue from one aquarium-kept harbor seal that died of a noninfectious condition served as a negative control.…”

“…Extraction of RNA and RT-PCR were performed as previously described (Stanton et al, 2002). Briefly, total RNA was isolated from FFPE blocks containing one or more tissues from a single animal by deparaffinization, phe- Amplicons were separated by agarose gel electrophoresis and visualized by ethidium bromide staining and ultraviolet transillumination.…”

“…Bands of the appropriate molecular mass were excised from gels, and DNA was extracted (Qiagen Gel Extraction Kit, Valencia, California) and subjected to direct sequencing by oligonucleotide-directed dideoxynucleotide chain termination cycle sequencing reactions. Betaactin mRNA served as a control for the extraction of amplifiable RNA from tissue blocks, as previously described (Stanton et al, 2002).…”

A Suspected Canine Distemper Epidemic as the Cause of a Catastrophic Decline in Santa Catalina Island Foxes (Urocyon Littoralis Catalinae)

“…A semi-nested reverse-transcription polymerase chain reaction (PCR) for a 149-basepair fragment of the canine distemper virus phosphoprotein gene was conducted on formalin-fixed paraffin-embedded sections of lung, using previously described methods (Stanton et al, 2002). Each reaction step was conducted using appropriate negative and positive controls.…”

Retrospective Serological Analysis of Spontaneous CDV Infection in 192 Dogs

“…For the diagnosis of CDV infection, viral RNA, viral protein and antibody were examined by PCR, immunohistochemistry and serological testing [6,14].…”