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 ...
(USA), researchers observed a hand-reared white tern hatchhing (Gygis alba rothschildi) develop vesicular lesions on the webbing between its toes, 6 days after falling out of its nest. Vesicular fluid collected from the foot lesions contained virus-like particles having typical calicivirus morphology. Cahicivirus RNA was detected in the vesicular fluid by dot hybridization with a group-specific calicivirus copy DNA probe. Attempts to cultivate the virus in African green monkey kidney cells and porcine kidney cells were unsuccessful. This is the first report of a cahicivirus infection associated with vesicular disease in a wild avian species.
Formalin-fixed paraffin-embedded tissues from one Caspian seal (Phoca caspica), one harp seal (Phoca groenlandica), one hooded seal (Cystophora cristata), and one harbor seal (Phoca vitulina vitulina) were used to compare the utility of immunohistochemistry (IHC) versus that of a novel seminested reverse transcriptase polymerase chain reaction (RT-PCR) to detect and differentiate canine distemper virus (CDV) and phocine distemper virus (PDV). Four antibodies made against PDV were able to detect both viruses. Two antibodies made against cetacean morbillivirus (CMV) did not label antigens from either CDV or PDV. A third anti-CMV antibody inconsistently stained CDV antigens but did not label PDV antigens. The seminested RT-PCR was able to detect RNA of the phosphoprotein gene in all positive cases. Nucleotide sequence analyses of seminested RT-PCR products were used to differentiate CDV RNA from PDV RNA. From these data, it was determined that IHC using antibodies generated against PDV provided a rapid means of detection for both CDV and PDV antigens; however, differentiation between CDV and PDV was achieved only with the RT-PCR assay.
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