Comparison of three in situ methods that targets nucleic acid to detect PCV2

Postweaning multisystemic wasting syndrome (PMWS) is still one of the major economic problems for the pig industry. To confirm PMWS, it is necessary to fulfil the following diagnostic criteria: 1) specific clinical and necropsy findings; 2) specific histologic lesion and 3) presence of moderate to significant amount of PCV2 by immunohistochemistry (IHC) or in situ hybridization (ISH). The fact that the virus is ubiquitous in pig populations worldwide, including healthy animals, makes PCR- or isolation-based methods non-specific for disease identification. Criterion 3 has a crucial value since it confirms PMWS, however PCV2 can be present in the host as a bystander with low virus loads; this renders ISH and IHC ineffective. The most reliable method for detecting virus under these circumstances is PCR, since the virus load is usually < 10⁸ copies of nucleic acid per g of total DNA. An even more sensitive variation on PCR-based detection of PCV2 and other swine viruses is in situ PCR (IS-PCR). Still rarely used in veterinary medicine, this technique offers tremendous potential.

For the study 10 paraffin blocks were selected, 2 originating from lymph nodes negative for PCV2 by PCR, 6 PCV2 positive lymph nodes by ISH and 2 fetal livers, negative by ISH and positive by PCR. Primers, cycling conditions and probes were designed to amplify and detect a 501bp region of PCV2 ORF1 as was previously described. For direct (d)IS-PCR amplicons were directly labeled with DIG. For indirect (i)IS-PCR and for ISH a 41 base length DIG probe was used. Cycling conditions were optimized for IS-PCR.

In all of the 6 positive lymph nodes the three methods showed positive result. However there was clear difference in the intensity of the signal. The lowest was obtained with ISH while the highest was in slides where dIS-PCR was applied. In two fetal livers, both dIS-PCR and iIS-PCR were able to detect PCV2, dIS-PCR seemed to be more sensitive while ISH stayed negative. Negative lymph nodes were negative by all methods.

IS-PCR seems to be very specific and versatile diagnostic tool more sensitive than ISH. IS-PCR was able to detect low number of copies of nucleic acid in tissues. However IS-PCR cannot completely replace ISH since it seems that is not suitable for detection when nucleic acid is present in high copy numbers.

Disclosure of Interest: None Declared.