Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

Appearance of slow-reacting and complement-requiring neutralizing antibody in cattle infected with Akabane virus

Slow-reacting complement-requiring neutralizing (NT) antibody was detected in sera from cattle 2 weeks after infection with Akabane virus. Bovine sera obtained 3 or 4 weeks after infection contained slow-reacting noncomplement-requiring NT antibody. The slow-reacting complement-requiring NT antibody was sensitive to 2-mercaptoethanol (2-ME), whereas the slow-reacting noncomplement-requiring NT antibody was resistant to 2-ME. The initial phase may represent the IgM response and the later phase a change to IgG. A NT test was developed in which virus-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 60 min; this gave an improved sensitivity over the previous incubation at 37 degrees C for 60 min.     

Energy metabolism in canine erythrocytes associated with inherited high Na+- and K+-stimulated adenosine triphosphatase activity

Energy metabolism in canine erythrocytes associated with inherited high Na+- and K+-stimulated adenosine triphosphatase [(Na,K)-ATPase] activity (HK cells) was compared with that in normal canine erythrocytes (LK cells). Activities of some of the glycolytic enzymes in the HK cells were significantly higher than those in LK cells. The concentrations of adenosine triphosphate (ATP) and glycolytic intermediates in HK cells were almost equal to those in LK cells. Glucose utilization and lactate production by HK cells in vitro and incorporation of [32P]orthophosphate or [14C]glucose into 2,3-diphosphoglycerate in HK cells were higher than in LK cells. Radioactivity of [32P]ATP in HK cells was lower than in LK cells, but increased to approximately that of LK cells when (Na,K)-ATPase of HK cells was completely blocked by ouabain. When HK cells and LK cells were incubated in the absence of glucose, the concentration of ATP in HK cells was decreased more than that of LK cells. Although ouabain reduced the rate of decrease in ATP in HK cells, the decrease in ATP in HK cells was still 2-fold that in LK cells. The half-life of HK cells was about one-half that of LK cells. The results indicated that glycolysis is greater in HK cells than in LK cells, and that the increased glycolysis in HK cells was stimulated by an increased rate of ATP breakdown for active cation transport by the (Na,K)-ATPase and by increased degradation of ATP for some other pathway, eg, glutathione synthesis. Thus, the increased demand for ATP in HK cells might result in shortening the lifespan of HK erythrocytes.     

Effect of group size and feeder type on growth performance and feeding patterns in finishing pigs

The effects of four group sizes (2, 4, 8, and 12 pigs per pen) and two single-space feeder types (conventional and electronic feed intake recording equipment [FIRE]) on feed intake, growth performance, and feeding patterns were determined in 208 crossbred finishing pigs (equal numbers of barrows and gilts) between 84.4 (SD = 0.81) to 112.8 (SD = 1.08) kg BW over a 4-wk period. Pigs were given ad libitum access to a corn-soybean meal-based diet (15.9% CP; 0.79% lysine; 3,328 kcal ME/kg). The floor space allowance was 0.9 m2/pig for all treatments. Growth rates were not different for the two feeder types; however, feed intake was lower and gain:feed ratio higher for pigs on the FIRE feeders (P < 0.01). Feed intake, growth rate, and gain:feed ratio were not different (P > 0.05) among the group sizes. Number of feeder visits per day decreased and feed intake per visit, feeder occupation time per visit, feed consumption rate, and percentage of time the feeder was occupied increased with group size (P < 0.05). Feed intake per visit had the strongest correlation with daily feed intake (r = 0.54; P < 0.01) and was negatively correlated with gain:feed ratio (r = -0.38; P < 0.01). However, the correlations between growth performance and other feeding pattern traits were relatively weak (r < or = 0.30). As group size increased, diurnal variation in number of feeder visits and feed consumed per hour decreased. There was no difference in time spent sitting and standing between the two feeder types. The proportion of time spent eating was generally lower for the larger groups on both feeders. The proportion of time spent lying was similar across group sizes for pigs on the conventional feeders but was greater for pigs in the larger groups on the FIRE feeders. This study suggests that finishing pigs can maintain feed intake and growth rate by changing feeding behavior as group size increases from 2 to 12.     

Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas

Pituitary-dependent hyperadrenocorticism (PDH) caused by pituitary corticotroph adenoma is a common endocrine disorder in dogs. The ratio between pituitary height and the area of the brain (P/B) has been used to evaluate the pituitary size. A P/B ratio > 0.31 indicates an enlarged pituitary, whereas a P/B ratio ≤ 0.31 indicates a nonenlarged pituitary. The aim of this study was to investigate the expression of proliferation markers Ki-67 and minichromosome maintenance-7 (MCM7) in canine corticotroph adenomas in enlarged and in nonenlarged pituitaries and to evaluate their relation with the size of canine pituitary corticotroph adenomas. Ki-67 and MCM7 expression in ACTH-positive tumor cells was determined by dual-labeling immunohistochemistry in resected corticotroph adenomas from 15 dogs with PDH. The mean ± SD Ki-67 labeling index (LI) was 0.55% ± 0.59% in corticotroph adenomas with nonenlarged pituitaries and 1.6% ± 0.6% in adenomas with enlarged pituitaries. The MCM7 LI in corticotroph adenomas with nonenlarged pituitaries and in adenomas with enlarged pituitaries was 2.9% ± 2.2% and 10.9% ± 3.7%, respectively. The Ki-67 LI and MCM7 LI were significantly greater in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P < 0.01 and P < 0.01, respectively). The MCM7 LI was significantly greater than the Ki-67 LI in adenomas (P < 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r = 0.820, P < 0.01), and the P/B ratio was positively correlated with the Ki-67 LI (r = 0.560, P = 0.03) and the MCM7 LI (r = 0.854, P < 0.01). In conclusion, canine corticotroph adenomas in enlarged pituitaries show greater proliferation potential than do adenomas in nonenlarged pituitaries. MCM7 expression was significantly greater than Ki-67 expression in canine pituitary corticotroph adenomas. Thus, MCM7 may be superior to Ki-67 as a proliferation marker in pituitary tumors.     

Technical note: recombinant LHRH fusion protein suppresses estrus in heifers

A recombinant luteinizing hormone-releasing hormone (LHRH) fusion protein was evaluated for its effectiveness in suppression of estrus in heifers. Eight heifers were randomly assigned to two equal treatment groups. Treatments consisted of recombinant ovalbumin-LHRH-7 or recombinant ovalbumin (control). This recombinant chimeric fusion protein consisted of ovalbumin with seven LHRH peptides (ovalbumin-LHRH-7). The plasmid for this protein was expressed in E. coli and was collected and purified as an insoluble protein. One milligram of the respective proteins was suspended in 2 mL of Z-Max adjuvant and administered by intramammary injection three times at 7-wk intervals. Luteinizing hormone-releasing hormone antibody binding was elevated in heifers treated with ovalbumin-LHRH-7 compared to ovalbumin-treated heifers (P < .05). Serum progesterone concentrations (< 1 ng/mL) indicate that the estrous cycle of the four heifers treated with ovalbumin-LHRH-7 was suppressed for a time period ranging from 60 to 238 d, which was different from control heifers (P < .01). Serum progesterone for the control heifers continued to exhibit cyclic profiles over the experimental period. This preliminary study in heifers demonstrated that a chimeric LHRH fusion protein induced elevated concentrations of circulating LHRH antibodies that suppressed estrus for an average of 122 +/- 41 d.     

Antibodies produced by mice immunized with recombinant vaccinia viruses expressing two different types of a major Theileria sergenti surface antigen (p32) react with the native surface antigen.

A 32 kDa major surface antigen, p32, of Theileria sergenti at the piroplasm stage is the main target of the host immune response. The immunogenic property of the p32 varies in some strains among the population of Theileria sergenti in Japan where the Chitose type and the Ikeda type are the most common varieties. We have constructed vaccinia virus recombinants vv/p32C and vv/p32I which harbor the Chitose and Ikeda types of p32 gene, respectively. It was found that vv/p32C and vv/p32I produced type-specific p32 which did not cross react with the monoclonal antibodies (mAbs) against the other type of p32. When mice were immunized with vv/p32C and vv/p32I, antibodies against p32 were detectable 2 weeks after the immunization, and these antibodies reacted with the native surface antigen in purified T. sergenti merozoite.