Effects of fixative type and fixation time on the detection of Maedi Visna virus by PCR and immunohistochemistry in paraffin-embedded ovine lung samples.
In doubtful cases, the histopathological diagnosis of lesions induced by Maedi Visna virus (MVV), a chronic multisystemic lentiviral disease of sheep, needs to be confirmed by the demonstration of MVV in the tissues. The influence of fixatives and the duration of fixation on the detection of MVV by immunohistochemistry (IHC) and PCR in paraffin-embedded tissues was assessed in lung samples with lesions in different degree, from five sheep serologically positive. Samples were fixed in 10% neutral buffered formalin (NBF), Bouin's solution (BS) and a zinc salts-based fixative (ZSF), for different periods of time between 24 h and 30 days. The three fixatives preserved the morphology of the tissues, although in ZSF-fixed samples an increase in the number of desquamated cells was seen in the alveoli. Tissues showed a similar degree of immunolabelling, irrespective of the duration of fixation using ZSF and NBF fixatives. MVV nucleic acids could be detected in samples fixed up to 14 days in NBF and 30 days in ZSF. However, in BS fixed tissues, immunostaining was weak and non-specific signals were observed after 4 days of fixation. Amplification of proviral DNA could not be obtained by PCR in these samples. IHC detected viral antigens in all sheep whereas one sheep with mild lesions was always negative by PCR.
Benavides
Julio
J
Departamento de Patología Animal: Medicina Animal (Anatomía Patológica), Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain.
García-Pariente
Carlos
C
Gelmetti
Daniela
D
Fuertes
Miguel
M
Ferreras
María Carmen
MC
García-Marín
Juan Francisco
JF
Pérez
Valentín
V
D019342:Acetic Acid; D000818:Animals; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005404:Fixatives; D005557:Formaldehyde; D007120:Immunochemistry; D008168:Lung / Q000821:virology*; D016612:Paraffin Embedding; D010851:Picrates; D011021:Pneumonia, Progressive Interstitial, of Sheep / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction; D012756:Sheep; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology; D014790:Visna-maedi virus / Q000302:isolation & purification*; D017967:Zinc Compounds
D004279:DNA, Viral; D005404:Fixatives; D010851:Picrates; D014764:Viral Proteins; D017967:Zinc Compounds; D005557:Formaldehyde; C026239:Bouin's solution; D019342:Acetic Acid