Srečko Koren

Trophoblastic Interferons: Do they Modulate Uterine Cellular Markers at the Time of Conceptus Attachment in the Pig?

Between days 12 and 20 of pregnancy, the trophectoderm of the porcine conceptus secretes two species of interferons (IFN): IFN-gamma (Type II), which is produced in substantial amounts, and IFN-delta (type I), for which secretion peaks at days 15-16 of gestation. The role of these embryonic IFNs is not known. We made the assumption that, in the pig, one possible role of these IFNs may be the remodelling and/or depolarization of the uterine endometrial epithelium, as a prerequisite for implantation and establishment of a functional placenta. A comparative analysis by immunohistochemistry of several cell membrane markers and ECM components of the cyclic and pregnant uterus was performed at day 15 post-oestrus. The markers were those likely to differ between a pregnant and cyclic uterus, or between different stages of pregnancy. A highly specific marker of IFN-gamma activity, namely MHC class II antigens in the uterine mucosa, was also examined. This study provides so far unreported data: in the endometrial epithelium of the pregnant uterus, we observed a partial relocalization of ZO-1, a marker of epithelial tight junctions, thus suggesting significant changes to the endometrial polarity. Heparan-Sulphate Proteoglycan (HSPG) expression did not differ significantly between cyclic and pregnant uteri. In contrast with the accepted rodent model of trophoblast-uterus adhesion, the porcine trophoblast and luminal epithelium were negative for HSPG. Finally, MHC class II antigens were absent from the cyclic uterus, but markedly induced in the day 15 pregnant uterus, particularly in endothelial cells, suggesting that IFN-gamma may indeed cross the maternal epithelium. This hypothesis was supported by the observation of IFN-gamma immunoreactivity associated with clusters of endometrial cells in the pregnant uterus.

Detection and molecular characterisation of noroviruses and sapoviruses in asymptomatic swine and cattle in Slovenian farms

Acute infectious caliciviral gastroenteritis is a common illness in people all over the world. Two genera of the Caliciviridae family, Norovirus and Sapovirus, which usually cause disease in humans, can also be found in animals where they do not always cause clinical signs of gastroenteritis. To investigate the presence of norovirus (NoV) and sapovirus (SaV) strains in asymptomatic swine and cattle, a total of 525 faecal (406 pigs and 119 cattle) specimens were collected during 2004 and 2005 from 8 pig and 4 cattle farms geographically dispersed across Slovenia. RT-PCRs and sequencing were carried out using primers targeting RdRp and capsid regions of both NoVs and SaVs. NoV positivity was detected in both bovine (2/108 [1.9%]) and porcine (5/406 [1.2%]) faecal specimens while SaV positivity was present only in porcine (29/406 [7.1%]) specimens. All porcine NoV strains (n=5) detected were attributed to a single farm, while the porcine SaV strains (n=29) detected came from 5 different farms. Phylogenetic analysis of nucleotide sequences of partial RdRp fragments placed two of the bovine NoV strains in genogroup GIII. Of the 5 porcine NoV strains, 4 clustered with GII.11, while 1 strain showed the presence of GII.18. The majority [24/29, 82.7%] of the porcine SaV strains clustered in GIII within two separate lineages, while 5 strains clustered into recently identified genetic clusters GVII (3 strains), GVIII (1 strain) and unknown (1 strain), respectively. Although NoV and SaV strains in asymptomatic swine and cattle were detected at low levels, they were still phylogenetically placed in a common pattern within both genera showing great genetic variability. There were no detected human-like strains in this study.

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Tetracycline-controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon-gamma in the RK-13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon-gamma (rGPoIFN-gamma) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN-gamma cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN-gamma (up to 16 microg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of 35S-Methionine, labelled rGPoIFN-gamma with natural leukocytic IFN-gamma after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN-gamma species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N-glycosydase F. The biological activity of rGPoIFN-gamma was in the same range as that of natural leukocytic PoIFN-gamma (2 x 10(6) U/mg). Eventually, this recombinant mammalian IFN-gamma should constitute a very useful substitute for leukocyte PoIFN-gamma in in vitro or in vivo experiments.

Multiplication of HVT FC-126 (Herpesvirus turkey) virus in the kidney cell lines of no avian origin

The presented experiments were aimed to cultivate and multiplicate HVT FC-126 in the PLA (Adult pig kidney) and GL-4 (Gerbil kidney) cell lines. Two different HVT FC-126 vaccine strains were used: Marikal SPF (Veterina d.o.o., Croatia) and Lyomarex (Merial, USA). They were adapted to the PLA and GL-4 cell lines. After adaptation, they were titrated on PLA (TCID50 2^4.23) and GL-4 (TCID50 2^4.96). On both cell lines they show similar CPE (cytophatic effect). The difference between them was detected using Real Time PCR, which was also positive by agarose gel analysis for the virus contained in Lyomarex, but not in the Marikal SPF. It can be concluded that both cell lines are sensitive to HVT FC-126 and the virus can be multiplied in high titers though much lower than in the calf intestinal epithelial cell line (CIEB) cells (TCID50 2^7.93).