A genome-wide significant association on chromosome 15 for congenital entropion in Swiss White Alpine sheep.

Entropion is a known congenital disorder in sheep presumed to be heritable but no causative genetic variant has been reported. Affected lambs show a variable inward rolling of the lower eyelids leading to blindness in severe cases. In Switzerland, the Swiss White Alpine (SWA) breed showed a significantly higher prevalence for entropion than other breeds. A GWAS using 150 SWA sheep (90 affected lambs and 60 controls), based on 600k SNP data, revealed a genome-wide significant signal on chromosome 15. The 0.2 Mb associated region contains functional candidate genes, SMTNL1 and CTNND1. Pathogenic variants in human CTNND1 cause blepharocheilodontic syndrome 2, a rare disorder including eyelid anomalies, and SMTNL1 regulates contraction and relaxation of skeletal and smooth muscle. WGS of a single entropion-affected lamb revealed two private missense variants in SMTNL1 and CTNND1. Subsequent genotyping of both variants in 231 phenotyped SWA sheep was performed. The SMTNL1 variant p.(Asp452Asn) affects an evolutionary conserved residue within an important domain and represents a rare allele, which occurred also in controls. The p.(Glu943Lys) variant in CTNND1 represents a common variant unlikely to cause entropion as the mutant allele occurred more frequently in non-affected sheep. Therefore, we propose that these protein-changing variants are unlikely to explain the phenotype. Additionally, WGS of three further disconcordant pairs of full siblings was carried out but revealed no obvious causative variant. Finally, we conclude that entropion represents a more complex disease caused by different non-coding regulatory variants.

Elimination of virulent strains (aprV2) of Dichelobacter nodosus from feet of 28 Swiss sheep flocks: A proof of concept study.

Virulent ovine footrot caused by Dichelobacter nodosus is an endemic disease worldwide. It is associated with severe pain, impaired animal welfare and economic losses. The competitive real-time PCR for the differentiation of virulent aprV2 and benign aprB2 strains of Dichelobacter nodosus provides an objective, rapid and sensitive diagnostic tool for footrot surveillance, especially as it enables early detection of subclinical carriers of virulent strains. The aim of this study was to evaluate the feasibility of complete elimination of Dichelobacter nodosus strains carrying the aprV2 gene from sheep flocks. The treatment protocol was based on careful removal of loose and severely overgrown claw horn, weekly stand-in foot baths of the entire flock for 10 min per sheep, using a 10% zinc sulphate disinfectant solution, clinical and PCR follow-up and isolation or culling of non-responders. Dichelobacter nodosus strains carrying the aprV2 gene were successfully eliminated from the feet of the sheep of all 28 flocks (100%) participating in the study within 6-19 weeks of weekly foot bathing. A strong correlation between the length of time for weekly foot bathing to eliminate the virulent strains and the within-flock prevalence of clinical footrot at the beginning was observed (rho, 0.68; P <0.001). A statistically significant correlation was not detected between flock size and the length of time for weekly foot bathing (rho, 0.28; P = 0.14), or the prevalence of clinical footrot at study commencement (rho, -0.04; P = 0.82), respectively. In conclusion, a complete elimination of Dichelobacter nodosus strains carrying the aprV2 gene in sheep flocks was possible with a protocol based on careful trimming, weekly stand-in foot baths, and identification of infection using a specific PCR-test and isolation or culling of non-responders.

The porcine follitropin receptor: cDNA cloning, functional expression and chromosomal localization of the gene.

The porcine follitropin receptor-encoding cDNA (pFSHR) was cloned using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from porcine granulosa cells was used as template. Two overlapping cDNA fragments encoding, respectively, aa 1 to 290 and aa 191 to 694 of the pFSHR were obtained. Taken together, the two fragments represented the whole coding sequence, assuming a comparable length for the FSHR from the porcine, rat and human species. Functionality of the cloned receptor was assessed by expression experiments; COS cells transfected with the pFSHR cDNA exhibited high-affinity specific binding for [125I]hFSH and FSH-dependent cAMP production. The primary sequence of the porcine FSHR N-terminal hormone-binding domain showed high percentages of identity with the sequences from ovine, human, and rat origins. A truncated form of the pFSHR cDNA, lacking aa 75 to 124 in the N-terminal domain, was also cloned and sequenced. A PCR-derived cDNA fragment of 1.45 kb was used as gene-specific hybridisation probe to map the pFSHR-encoding gene by radioactive in situ hybridization. This gene was found co-localized (as in human) with the porcine lutropin hormone receptor (pLHR)-encoding gene on the q2.2-q2.3 region of pig chromosome 3.

Generation of virus specific cytotoxic T cells in vitro. III. Spleen cells stimulated by viral antigens generate alloreactive cytotoxic T cells.

Cytotoxic T lymphocytes (CTL) from DBA/2 strain mice primed with Sendai virus (SV) in vivo were activated by secondary stimulation of spleen cells with viral antigens in vitro and analyzed for their target antigen specificity. These effector cells lysed syngeneic Sendai virus infected target cells, marginally a variety of non-infected targets and had a strong cytotoxic effect on H-2b targets. Studies on the antigenic requirements revealed that all SV preparations which generated specific CTL also induced the alloreactive populations. Similar results were found in the response to Newcastle disease virus (NDV) and some influenza A viruses; all these viruses were mitogenic for lymphocytes. Experiments on the cellular requirements indicated that virus specific and alloreactive cells can be separated by their requirements for help and for restimulation. By competition experiments both activities could be attributed to clearly separable T cell subpopulations. The induction mechanism of alloreactive T cells by viral antigens is discussed.

Beneficial effect of nifedipine and moxonidine on glomerulosclerosis in spontaneously hypertensive rats. A micromorphometric study.

The effect of calcium channel blockers on the development of glomerulosclerosis and progression of renal failure in different models of renal injury is still controversial. We compared the effects of blood pressure lowering with high doses of nifedipine (27 mg/kg body weight/day) and with the sympatholytic agent moxonidine (8 mg/kg body weight/day) in 6-month-old male spontaneously hypertensive rats (SHRsp). As controls we studied untreated hypertensive SHRsp and normotensive Wisfar-Kyoto rats (WKY). After 3 months of treatment, left ventricular (LV) weight and systolic blood pressure (tail plethysmography) were lower in both treated groups (144 +/- 21.4 mm Hg and 144 +/- 13.5 mm Hg v 193 +/- 38.6 mm Hg in untreated SHRsp), but remained higher than in WKY (116 +/- 16.0 mm Hg). Stereological analysis of perfusion fixed kidneys showed an unchanged total volume of cortex and medulla, but a higher mean glomerular volume in nifedipine treated SHRsp. The glomerulosclerosis index was similarly reduced by both antihypertensive agents (92.8 +/- 68.1 in untreated SHRsp v 27.2 +/- 12.9 and 18.2 +/- 9.8 in the two treatment groups, respectively). This was accompanied by a similar reduction of total cortical arterial wall volume (from 36.3 +/- 16.5 mm3 to 18.9 +/- 2.53 and 15.3 +/- 2.53 mm3, respectively) and by reduction of tubular atrophy or interstitial fibrosis, respectively. In this model nifedipine lowered blood pressure and inhibited development of glomerulosclerosis to the same extent as a sympatholytic agent. This was accompanied by increased glomerular volume and filtration area in nifedipine treated animals.

Effects of nifedipine and moxonidine on cardiac structure in spontaneously hypertensive rats. Stereological studies on myocytes, capillaries, arteries, and cardiac interstitium.

Light and electron microscopic stereological studies were performed on the myocardium of spontaneously hypertensive rats (SHR-SP) before and after treatment with nifedipine (27 mg/kg body weight/day) and the antisympathotonic agent moxonidine (8 mg/kg body weight/day). The treated groups were compared with nontreated SHR-SP and normotensive WKY (n = 10 in each group). At the beginning of therapy (when the male SHR-SP were 6 months old), blood pressure was increased and left ventricular hypertrophy had developed whereas pathologic changes of myocardial structure were not observed. After 3 months, the nontreated hypertensive rats showed cardiac fibrosis, activation and proliferation of interstitial cells, wall thickening of intramyocardial arteries, reduced capillarization as well as focal degeneration of myocytes at the ultrastructural level. Both treatments showed similar effects on blood pressure, degree of hypertrophy, and cardiac structure. Blood pressure as well as the degree of hypertrophy were significantly reduced. As far as myocardial fibrosis, capillarization, and regressive changes of myocytes are concerned a complete normalization was observed. Furthermore, nifedipine enhanced capillary supply beyond the normal level by induction of capillary neoformation. Microarteriopathy and activation of nonvascular interstitial cells (first step in development of interstitial myocardial fibrosis) were significantly suppressed by therapy, but the level of the normotensive control could not be maintained. Additional experiments with a low dose combination therapy of nifedipine and moxonidine that did not reduce blood pressure provided evidence that hypertension is an important determinant of the alterations of intramyocardial arteries, but not of cardiac interstitial fibrosis.

Effects of nifedipine and moxonidine on cardiac structure in spontaneously hypertensive rats (SHR)--stereological studies on myocytes, capillaries, arteries, and cardiac interstitium.

Light and electron microscopic stereological studies were performed on the myocardium of spontaneously hypertensive rats (SHR) before and after treatment with nifedipine (27 mg/kg b.w./day) and the sympatholytic agent moxonidine (8 mg/kg b.w./day). The treated groups were compared with nontreated SHR and normotensive WKY (n = 10 in each group). When the therapy was started in 6-month old male SHR, blood pressure was increased and left ventricular hypertrophy had developed. On the other hand, pathologic changes of myocardial structure were not observed. After 3 months, the nontreated hypertensive rats showed cardiac fibrosis (volume density of fibrosis + 45%), activation and proliferation of interstitial cells (volume density of nonvascular interstitium + 240%), media hypertrophy of small arteries (total volume of arterial media in the left ventricle + 180%), reduced capillarization (length density of capillaries--11%), as well as focal degeneration of myocytes at the ultrastructural level. Both treatments showed similar effects on blood pressure, degree of hypertrophy, and cardiac structure. Blood pressure as well as degree of hypertrophy were significantly reduced (relative left ventricular weights: --25% and --16%). As far as myocardial fibrosis, capillarization, and regressive changes of myocytes are concerned a complete normalization was observed. Microarteriopathy and activation of nonvascular interstitial cells (first step in development of interstitial myocardial fibrosis) were significantly suppressed by therapy (total media volume --40%, volume density of nonvascular interstitium --38%), but the normal level of the normotensive control could not be maintained (+ 70%, + 111% vs WKY). This may be due to the slightly elevated systolic blood pressure despite therapy (+ 25%, vs WKY) or to hormonal factors in SHR which are independent of blood pressure. Since nifedipine and moxonidine are pharmacologically different drugs with different effects on sympathetic activity, one may cautiously conclude that increase in blood pressure itself is an important determinant of arterial, interstitial as well as myocellular alterations which are related to the pathogenesis of hypertensive heart muscle disease.