Spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus.

The spike glycoprotein of infectious bronchitis virus (IBV), a coronavirus, is translated as a precursor protein (So), then cleaved into two subunits (S1 and S2) by host cell serine proteases. In this study, we compared the cleavage recognition site of 55 IBV isolates to determine if the cleavage recognition site sequence, which consists of five basic amino acid residues, correlates with host cell range, serotype, geographic origin, and pathogenicity as it does in orthomyxoviruses and paramyxoviruses. The most common cleavage recognition site observed (33 of 55 viruses) was Arg-Arg-Ser-Arg-Arg, representing at least 11 different serotypes. Thus, cleavage recognition site does not appear to correlate with serotype. We also determined that cleavage recognition site sequence does not correlate with pathogenicity because attenuated and pathogenic isolates (different passages of the same virus) contain identical cleavage recognition site sequences. In addition, nephropathogenic strains had the same cleavage recognition site sequence as many nonnephropathogenic isolates. Cleavage recognition site sequence does correlate with viruses in different geographic regions, which may be an important characteristic to examine in epidemiologic studies. An IBV monoclonal antibody neutralization-resistant mutant (NR 18) had an unusual substitution of Ile for Arg at the fourth position, giving the sequence Arg-Arg-Ser-Ile-Arg, which likely prevents cleavage and, thus, destroys the conformationally dependent monoclonal antibody binding epitope. Six residues on the amino-terminal side of the cleavage recognition site are conserved in 31% of the isolates and consist of only one or two basic amino acids. Thus, the number of basic residues around the cleavage recognition site does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with envelope glycoproteins in orthomyxoviruses and paramyxoviruses.     

Multiplex polymerase chain reaction for distinguishing Taylorella equigenitalis from Taylorella equigenitalis-like organisms.

It is difficult to distinguish isolates of Taylorella equigenitalis, the cause of contagious equine metritis, from a T. equigenitalis-like organism isolated from asymptomatic donkeys and horses. Although T. equigenitalis is responsible for a severe, contagious disease of the reproductive tract of equids, the T. equigenitalis-like organism, although contagious, does not appear to produce disease. Because of the economic consequences of correctly distinguishing isolates of these 2 microorganisms, a polymerase chain reaction (PCR)-based assay was developed that will distinguish isolates of T. equigenitalis from the T. equigenitalis-like microorganism. The primers used in the PCR assay were designed to amplify unique regions of the gene encoding the 16S ribosomal RNA.     

Immunohistochemical demonstration of Leu-7 (HNK-1), Neurone-specific Enolase (NSE) and Protein-Gene Peptide (PGP) 9.5 in the developing camel (Camelus dromedarius) heart

The development of the heart-conducting system has been controversially discussed. The common opinion that these specialized myocytes originate from mesodermal precursors has been challenged when nerve-specific antigens (Leu-7, NF, GIN2) were demonstrated in embryonic hearts of various species, suggesting a neural crest contribution to the embryonic conducting tissue. Anti-Leu-7 (HNK-1) antibodies were reported to reliably mark the conducting system in developing rat, chicken and human hearts. The present investigation was carried out on the hearts of 15 camel fetuses at 35, 45, 60, 75 and 100 cm crown-rump length (three specimens for each stage), in addition to three adult hearts. We investigated the antigenicity of cardiac structures for Leu-7, NSE (Neurone specific Enolase) and PGP (Protein Gene Peptide) 9.5. In all specimens investigated, both NSE and PGP 9.5 were expressed by cardiac nerves and conducting system components. The sinuatrial and atrioventricular nodes, the atrioventricular bundle as well as subendocardial and intramyocardial Purkinje fibers were stained. In contrast, the developing conducting system did not react with anti-Leu-7 antibody, although Leu-7 antigenicity was strongly expressed by the developing cardiac nerves. In adult camel hearts, the same pattern of immunoreactivity for the markers studied was still retained. Our results show that the expression of marker proteins for the developing conducting system is species-specific. Therefore, these markers are of little significance in discussions on the possible neurogenic nature of the heart conducting tissue.     

Growth and development of the digestive organs and some enzymes in broiler chicks after hatching.

1. Body weight and the weight of the digestive organs and activities of some digestive enzymes were determined from hatching to 23 d of age. 2. Relative daily growth rate peaked at 11 d of age (22% gain/d) and then decreased gradually. 3. The vitelline residue was decreased rapidly from 4.6 g at hatching to negligible values from 4 d of age. 4. Maximal allometric growth of the pancreas and small intestine was 4-fold and that of liver 2-fold greater than that of the body. 5. Activities (units/kg body weight) of the digestive enzymes measured in the pancreas and intestinal contents increased with age. In the pancreas maximal values were attained on day 8 for amylase and lipase and 11 for trypsin and chymotrypsin. In the small intestine maxima were attained on day 4 for lipase, 11 for trypsin and chymotrypsin and 17 for amylase. 6. The development of secretion of digestive enzymes in the post-hatched chick could be a limiting factor in digestion and subsequently in food intake and growth.     

Effects of transforming growth factor-α on parathyroid hormone- and parathyroid hormone-related protein- mediated bone resorption and adenylate cyclase stimulation in vitro

The effects of transforming growth factor-alpha (TGF alpha) were determined on the ability of parathyroid hormone (PTH) or parathyroid hormone-related protein (PTHrP) to stimulate bone resorption and adenylate cyclase in vitro. Bovine PTH-(1-34) and human PTHrP-(1-34) were equipotent in their ability to stimulate bone resorption in neonatal mouse calvaria with maximal stimulation (2.9 and 2.8-fold increases in 45Ca release, respectively) at a concentration of 10 nM. Combinations of TGF alpha with bPTH-(1-34) or hPTHrP-(1-34) had additive effects on their ability to stimulate bone resorption when submaximal concentrations of the agonists were used. There was no evidence of synergism between TGF alpha bPTH-(1-34) or hPTHrP-(1-34) in their ability to stimulate bone resorption in vitro, nor was TGF alpha able to increase bone resorption induced by maximal concentrations of bPTH-(1-34) or hPTHrP-(1-34). TGF alpha potentiated the effects of either bPTH-(1-34) or hPTHrP-(1-34) on the stimulation of adenylate cyclase in osteoblast-like ROS 17/2.8 cells. These data indicate that TGF alpha has additive effects with submaximal concentrations of PTH or PTHrP on their ability to stimulate bone resorption which may be important in the pathogenesis of humoral hypercalcemia of malignancy.     

Immune response in pigs to Aujeszky's disease viruses defective in glycoprotein g1 or gX.

Two Aujeszky's disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszky's virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszky's disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.     

One-step purification of the major rainbow trout immunoglobulin

Salmonid immunoglobulin purification has been hampered by time-consuming, labour-intensive, conventional chromatographic procedures. In this report we describe the use of immunoaffinity chromatography for the purification of immunoglobulins from trout serum in a single step. An anti-heavy chain monoclonal antibody (1G7) which reacts with more than 85% of total trout immunoglobulins was used to purify the trout immunoglobulin. The purity achieved was higher than 95%, and the immunoglobulins recovered were fully immunogenic. This method served equally well in isolating immunoglobulins from the sera of other salmonids (coho salmon and chinook salmon). The procedure should be useful in the standardization of salmonid immunoglobulin reagents.     

Genetic correlations among reproductive traits and uterine dimensions in mice

The objective of this experiment was to identify relationships among reproductive and uterine traits in mice having normal or crowded uterine conditions. Littermate females were randomly assigned to be either unilaterally ovariectomized (ULO) or to remain intact (C) and to be killed either 3 d after mating (PM) or 4 d after parturition (PP) in a 2 x 2 factorial design. Measurements taken were ovulation rate (OR) and uterine length (UL), wet weight (UWW), dry weight (UDW) and displacement (UDP) in PM females and number born (NB) and implantation rate (IMP) in PP females. Heritability estimates from full-sib correlations were .18, .01, .33, .04, .14, .47 and .06 for OR, IMP, NB, UL, UWW, UDW and UDP, respectively. Phenotypic correlations among uterine measurements were moderate to high and positive. Genetic correlations for C and ULO females for OR with NB were .62 and .73, respectively. Genetic correlations between C and ULO females were .53 for NB and 1.05 for OR. Genetic correlations of UL and UWW with NB were high for C (.70 and .59, respectively) and moderate for ULO (.47 and .36, respectively). Genetic correlations between NB and other uterine dimensions were lower.     

Canine distemper viral antigens and antibodies in dogs with rheumatoid arthritis

Dogs with canine rheumatoid arthritis had significantly elevated levels of antibodies to canine distemper virus. This increase was particularly seen in the synovial fluids, compared with paired sera, and was not found in dogs with infective arthropathies, osteoarthritis or in osteoarthritis secondary to rupture of the cranial cruciate ligament. Analysis of the immune complexes precipitated from synovial fluids showed immunoglobulins in all types of arthropathy. Western blotting analyses showed reactivity with anti-distemper antisera in immune complexes from dogs with rheumatoid arthritis, but not in immune complexes from dogs with other joint diseases. These results suggest that there are increased immune responses to distemper in canine arthritis and that these may be due to the presence of this paramyxovirus in affected joints. The implications for the role of a possible infectious agent in rheumatoid arthritis in the dog are considerable.     

Ultrasonic, needle, and carcass measurements for predicting chemical composition of lamb carcasses

Three groups (n = 147) of New Zealand mixed breed lambs averaging 170 d of age and 31.7 kg in weight were killed after a diet of pasture to determine whether the total depth of soft tissues over the 12th rib 11 cm from the dorsal midline (GR) could be measured in live lambs with sufficient accuracy to warrant its use as a selection tool for breeding flock replacements. Relationships among live and carcass measurements and carcass chemical composition also were determined. An ultrasonic measurement of GR in the live lambs was a more accurate predictor of carcass GR (r = .87) and percentage carcass fat (r = .80) than was a measurement of GR made with a needle (r = .80 and .67, respectively). Both measurements were sufficiently accurate to permit culling of over-fat lambs from breeding flock replacement prospects. The best single indicator of percentage carcass fat (r = .87) was a shoulder fat measurement, followed closely by carcass GR (r = .85). Both were superior to USDA yield grade for estimating carcass chemical composition in these young, lightweight lambs. These two measurements also were most highly related to percentage carcass protein (r = -.78 and r = -.77, respectively). These results indicate possibilities for improving the method of evaluating the composition of U. S. lamb carcasses.