A double-antibody sandwich ELISA (DAS-ELISA) was developed for detection of avian influenza virus (AIV) antigen. A monoclonal antibody to the viral nucleoprotein (NP) was used to coat the ELISA plates. A direct DAS-ELISA and an indirect DAS-ELISA were evaluated. In the direct DAS-ELISA, monoclonal antibody to the AIV NP conjugated with horseradish peroxidase was used. The direct DAS-ELISA was evaluated for its sensitivity to detect purified NP; this procedure detected as little as 0.1 ng. In the indirect DAS-ELISA, rabbit NP antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobin were used as primary and secondary antibodies, respectively. The indirect DAS-ELISA was evaluated for its ability to detect the AIV antigen in tracheal and cloacal specimens from turkeys inoculated with AIV. Results of indirect DAS-ELISA were compared with those of conventional virus isolation. Percentage agreement between indirect DAS-ELISA and virus isolation in AIV-positive samples was found to be 76.1% and, in AIV-negative samples, it was found to be 82.1%. These results indicate that the DAS-ELISA might be a viable alternative to virus isolation because of its rapidity, compared with virus isolation.

A study was performed to determine whether experimentally induced bovine leukemia virus (BLV) infection in cattle could be detected earlier by use of polymerase chain reaction (PCR) amplification of genomic DNA extracted from leukocytes than by use of conventional agar-gel immunodiffusion (AGID). The PCR primers were designed to amplify a 375-basepair region of the proviral gag gene. Five cows were identified that were BLV-negative on the basis of AGID and PCR results. At day 0, these cows were inoculated IM with blood pooled from 3 naturally infected cows. Blood samples were taken on days 0, 1, and 7, and every 2 weeks thereafter until 3 months after inoculation. Three of the cows were BLV-positive by AGID test results 3 weeks after inoculation, and the remaining 2 seroconverted at 5 weeks. In contrast, all 5 cows were BLV-positive by PCR results 7 days after inoculation and remained positive for the duration of the study. Five cows that were BLV-positive by AGID test and PCR results on day 0 and from which samples were obtained at the same times as those from the other 5 cows, remained BLV-positive by results of both tests during the course of the study. Results indicate that under experimental conditions, BLV infection in cattle can be detected as much as 2 to 4 weeks earlier by use of PCR than by use of the AGID test.

Flow-volume loops generated from 6 Standardbreds at rest and during treadmill exercise were evaluated for their use in detecting upper airway obstruction. Tidal breathing flow-volume loops (TBFVL) were obtained from horses at rest and exercising at speeds corresponding to 75% of maximal heart rate and at maximal heart rate. The TBFVL were evaluated, using a pulmonary function computer; calculated indices describing airflow rate and expiratory-to-inspiratory airflow ratio for individual loops were determined. In addition to TBFVL indices, standard variables of upper airway function also were measured: peak airflow, peak pressure, and calculated inspiratory and expiratory impedances. Measurements were recorded before left recurrent laryngeal neurectomy (LRLN; baseline) and 14 days after surgically induced left laryngeal hemiplegia. When horses were at rest, TBFVL shape and indices describing the loop were highly variable. In contrast, in exercising horses, TBFVL shape was consistent and coefficients of variation of loop indices were less during exercise than at rest. After LRLN, TBFVL from exercising horses indicated marked inspiratory airflow limitation, while the expiratory airflow curve was preserved. Peak inspiratory flow rate and inspiratory flow at 50 and 25% of tidal volume decreased, and the ratio of peak expiratory to inspiratory airflow and that of midtidal volume expiratory and inspiratory airflow rates increased significantly (P < 0.05). Inspiratory impedance also increased after LRLN. Although in resting horses TBFVL were not a useful indicator of upper airway obstruction, examination of TBFVL from exercising horses allowed objective, specific, and repeatable detection of upper airway obstruction. The technique was noninvasive, rapid, and well tolerated by horses; thus, it is a potentially valuable clinical diagnostic test.

A method of extracting bacterial DNA from swine feces was developed and used in a molecular assay for the presence of ileal symbiont (IS) intracellularis, formerly known as the Campylobacter-like organism associated with swine with proliferative enteritis. Hybridization with a digoxigenin-labeled, IS intracellularis-specific probe detected the presence of IS intracellularis at a concentration of 10(7) organisms/g of feces. This method was sufficient to detect is intracellularis in the feces of swine with experimentally induced and naturally acquired infection. Results of the hybridization were in agreement with those from histologic postmortem examination.