Technical note: Development of an enzyme-linked immunosorbent assay for the determination of florfenicol and thiamphenicol in swine feed

One polyclonal antibody against florfenicol and thiamphenicol was produced and a competitive ELISA was developed for the detection of florfenicol and thiamphenicol in swine feed. The ELISA gave a 50% inhibiting concentration of 1.02 ng/mL for florfenicol. For swine feed fortified with 0.05 to 3.0 mg/kg, the interassay recoveries of florfenicol and thiamphenicol ranged from 86.4 to 118.6%, whereas intraassay recoveries of both drug ranged from 90.1 to 126.5% with less than 15% CV. Results obtained from HPLC-tandem mass spectrometry indicated this ELISA procedure could be used as a convenient method for rapid screening of florfenicol and thiamphenicol in swine feed.     

Coxsackievirus B4 myocarditis in an orangutan.

A 37-year-old female orangutan died at the zoological garden. Autopsy examination demonstrated severe coxsackievirus B4 myocarditis immunohistochemically as a cause of the death. Apoptosis of the cardiac muscle cells was observed using the TdT-mediated dUTP-biotin nick endo labeling method and was considered to play a role in the myocarditis. Congestion of the liver and both lungs due to cardiac failure was also observed. Coxsackievirus infection is found frequently in the Okinawan human population. The present orangutan's infection might have come from visitors who were allowed to go near the orangutan. Malignant tumors, severe suppurative infections, and intestinal parasite infections were not observed. Epstein-Barr virus DNA was detected in lymph nodes, but there was no Burkitt's lymphoma.     

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.     

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.     

Disease-induced alterations in plasma drug-binding proteins and their influence on drug binding percentages in dogs

Disease-induced variations of plasma albumin (ALB) and alpha 1-acid glycoprotein (AAG) levels were investigated in dogs. Lower ALB (sometimes > 50% reduction) and higher AAG (sometimes > 10-fold increase) levels were observed in dogs with various diseases. Drug binding was determined at therapeutic concentrations using normal, low-ALB and high-AAG dog plasma. The binding percentages of the ALB-binding drugs decreased in low-ALB plasma, resulting in a large increase in unbound drug, particularly for naproxen (a 13-fold increase). The binding percentages of all AAG-binding drugs investigated in this study increased in high-AAG plasma, resulting in a large decrease in unbound drug, particularly for quinidine (99% decrease). The fluctuation in the unbound fraction of drugs could affect their efficacy or could cause side-effects. Veterinary clinicians should monitor the ALB and AAG levels in the plasma of patients and correct dosage regimens according to these levels, where field conditions permit this, in order to ensure the proper usage of drugs with high affinity for ALB or AAG.