An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: cytochrome b PCR-RLFP analysis.

One of the major risks in cell culture laboratories is the misidentification and cross-contamination of cell lines. Several methods have been used to authenticate cell lines, including isoenzyme profiling, the test suggested by European Farmacopeia, which is performed at the Tissue Culture Centre in Brescia. However, this method displays several disadvantages, such as high variability and low reproducibility, and it is time consuming and requires high cell concentrations to be performed. Therefore, an alternative method has been developed to confirm the specie of origin of 27 different animal cell cultures. A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was optimized, based on the use of a pair of primers that anneal to a portion of the cytochrome b gene in all the species. The amplification product was digested with a panel of six restriction enzymes, and the pattern derived was resolved on 3% high-resolution agarose gel. For 23 species, this protocol produced a unique restriction pattern, and the origin of these animal cells resulted to be confirmed by this analysis. Furthermore, results indicate that cytochrome b PCR-RFLP was able to amplify target sequences using very low amounts of deoxyribonucleic acid (DNA). Its sensitivity in detecting interspecies, cross-contamination was comparable to that of isoenzyme analysis (contaminating DNA should represent at least 10% of the total DNA). For 4 of the 27 species (sheep, dog, Guinea pig, and Rhesus monkey) the observed pattern, even if highly reproducible, showed additional bands; for these species, specific PCR was also performed.

Characterization of the interferon-α response of pigs to the weaning stress.

The interferon (IFN)-α response of pigs to the stressing event of early weaning was investigated in a field trial. All the animals under study remained healthy and tested negative for common viral infections. However, a low-titered IFN-α response was detected in many sera by a bioassay on Madin-Darby bovine kidney (MDBK) cells on day +6 after weaning. Porcine IFN-α was unambiguously identified by a neutralization assay on a pool of IFN-α-positive sera. By gel filtration chromatography, the antiviral activity of sera on MDBK cells could be traced back to 3 components of apparent molecular mass 27/18/<14 kDa. Additional components of apparent molecular mass 58 and 41 kDa were revealed by ELISA in Nonidet P-40 lysates of peripheral blood mononuclear cells (PBMC). Also, many pigs tested positive in flow cytometry assays on PBMC for intracellular IFN-α. The expression of porcine IFN-α genes was investigated by reverse transcriptase (RT) real-time polymerase chain reaction at days -1, +6, and +12 with regard to weaning in PBMC of 9 piglets. On days -1 and +12, IFN A5, A6, A12, as well as (in fewer pigs) A1, A7, A11, and A2 genes were shown to be expressed. On the contrary, none of the above genes was expressed on day +6, when plenty of pig sera were IFN-α-positive. Our results indicate that weaning causes the release of IFN-α and the transient shut-off of the corresponding gene transcriptions in PBMC. Interestingly, only IFN A9 gene transcription was shown in vitro to be virus induction-dependent.

Comparative evaluation of PRRS virus infection in vaccinated and naïve pigs.

The purpose of this study was to evaluate the time-course of the immune response to a field Porcine Respiratory and Reproductive Syndrome virus (PRRSV) strain in PRRS-naïve, untreated pigs, as well as in four groups of age and breed-matched pigs injected with a live attenuated PRRS vaccine, its adjuvant, an inactivated PRRS vaccine and an irrelevant, inactivated Porcine Circovirus type 2 (PCV2) vaccine, respectively. PRRSV infection was confirmed in all groups by PCR and antibody assays. The antibody response measured by ELISA took place earlier in pigs injected with the live attenuated vaccine, which also developed a much stronger serum-neutralizing antibody response to the vaccine strain. Yet, no clear protection was evidenced in terms of viremia against the field virus strain, which showed 11.1% nucleotide divergence in ORF7 from the vaccine strain. In vitro, the interferon (IFN)-γ response to PRRSV was almost absent on PVD 60 in all groups under study, whereas the prevalence of interleukin (IL)-10 responses to PRRSV was fairly high in PCV2-vaccinated animals, only. Results indicate that distinct patterns of immune response to a field PRRSV strain can be recognized in PRRS-vaccinated and naïve pigs, which probably underlies fundamental differences in the development and differentiation of PRRSV-specific immune effector cells.