bSurface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1 ؊/؊ pigs followed the same course as in SIGLEC1 ؊/؉ and SIGLEC1 ؉/؉ littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.
The concentration of circulating pregnancy associated glycoproteins (PAGs) early in pregnancy may serve as markers to predict late embryonic mortality or fetal mortality in cattle. In this study, pregnancies were established in dairy cows, by either fixed-time AI (FTAI) or fixed-time embryo transfer (FTET) with in vitro produced embryos. Circulating PAGs were measured with different combinations of antibodies in either a laboratory-based ELISA or a commercial ELISA. For the in-house ELISA, three monoclonal 'trapping' antibodies (A6, J2, and L4) and two polyclonal 'detection' antisera (antibodies F2 or 45) were used to quantify PAGs in serum from the same cows. The different assays were identified as follows: 'Mix-45' (A6, J2, and L4 with 45), 'Mix-F2' (A6, J2, and L4 with F2), and 'L4-F2': (L4 with F2); the commercial assay was from IDEXX. Ovulation was synchronized and FTAI or FTET was performed on day 0 or 7, respectively. Ultrasound-based diagnosis of pregnancy and serum collections occurred on day 30. The proportion of cows that subsequently experienced pregnancy loss between days 30 and 60 was 23% (43 of 183) and 16% (21 of 131) for the FTAI or FTET groups, respectively. In the FTAI group, mean serum concentration of PAGs detected with Mix-45 was higher in cows that maintained pregnancy (9.2 ± 0.4 ng/ml; mean ± SEM) compared with cows that experienced pregnancy failure (3.9 ± 0.6 ng/ml) between day 30-60 (P < .001). However, there was no difference (P > .69) in circulating concentrations of PAGs between cows that experienced loss or survival between days 30 and 60 when Mix-F2 or L4-F2 were used in an in-house ELISA. Likewise, a commercial assay also did not result in measurable differences in PAG concentrations between those animals that experienced loss or survival. Following FTET, circulating concentrations of PAGs on day 30 were lower (P < .001) in cows that experienced pregnancy failure compared to cows that maintained pregnancy when the Mix-45 and the commercial assay were used, but not with the other antibody combinations. A receiver operating characteristic curve showed that only the Mix-45 antibody combination was predictive (95% accuracy) of pregnancy loss but not the other antibody combinations following FTAI. However, both Mix-45 and the commercial assay were predictive of losses following FTET. In summary, although multiple PAG assay formats have been shown to accurately detect pregnancy, the ability to predict embryo survival during early gestation appears to be antibody dependent.
Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n 5 22) or with KLH alone (n 5 9), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.6 6 2.2 of pregnancy. Those ewes immunized against PAGs (n 5 7), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.9 6 1.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n 5 15), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1 6 15.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.
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