Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.     

Treatment of dogs infected with Hepatozoon americanum: 53 cases (1989-1998)

OBJECTIVE: To determine clinical and pathologic findings before and after short-term (group 1) and long-term (group 2) treatment in dogs with Hepatozoon americanum infection. DESIGN: Retrospective study. ANIMALS: 53 dogs with H. americanum infection. PROCEDURE: Medical records of dogs that were treated for hepatozoonosis diagnosed on the basis of meront or merozoite stages in skeletal muscle were reviewed. RESULTS: Circulating gametocytes of H. americanum were identified in 12 of 53 dogs. Dogs were treated with various drugs, including toltrazuril, trimethoprim-sulfadiazine, clindamycin, pyrimethamine, and decoquinate. Mean WBC counts prior to treatment were 85,700 and 75,200 cells/microl in groups 1 and 2, respectively, and 1 month after initiation of treatment were 12,600 and 14,600 cells/microl, respectively. Initial response to treatment was excellent in all dogs. Twenty-three of 26 dogs in group 1 relapsed at least once and died within 2 years; mean (+/- SD) survival time was 12.6+/-2.2 months. Twenty-two of 27 group-2 dogs survived; 11 dogs had no clinical signs and were still receiving decoquinate (mean duration of treatment, 21 months), 11 dogs had no clinical signs after treatment for 14 months (range, 3 to 33 months; mean survival time, 39 months [range, 26 to 53 months]), 2 dogs were lost to follow-up, and 3 dogs were euthanatized because of severe disease. CONCLUSIONS AND CLINICAL RELEVANCE: Although no treatment effectively eliminated the tissue stages of H. americanum, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and excellent quality of life.     

Subchondral cystic lesions of the proximal extremity of the tibia in horses: 12 cases (1983-2000)

OBJECTIVE: To determine clinical and radiographic features of subchondral cystic lesions (SCL) of the proximal extremity of the tibia in horses that could be used to classify these lesions as being related to osteochondrosis or osteoarthritis and to evaluate results of surgical debridement. DESIGN: Retrospective study. ANIMALS: 12 horses with 14 SCL. PROCEDURE: Medical records and radiographs obtained before and after treatment were reviewed. RESULTS: In 6 young horses (8 lesions), SCL were considered to be related to osteochondrosis; all involved the lateral tibial condyle. The remaining 6 horses were mature and had radiographic evidence of osteoarthritis in addition to SCL. Arthroscopic debridement was performed in 4 horses in which lesions were considered to be a result of osteochondrosis and in 3 horses with osteoarthritis. Three horses in which SCL were considered to be a result of osteochondrosis performed athletically after debridement. Two horses with moderate osteoarthritis returned to work after arthroscopic debridement but at a lower level of athletic performance. One horse with SCL related to osteochondrosis responded to medical treatment and went on to race. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that arthroscopic debridement of SCL is feasible in horses in which lesions involve the cranial portion of the lateral or medial tibial condyle, and that treated horses may be able to perform athletically.     

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.     

Apparent ileal digestibility of amino acids and the digestible and metabolizable energy content of dry extruded-expelled soybean meal and its effects on growth performance of pigs

We conducted three experiments to determine the apparent ileal digestibility of amino acids (Exp. 1), metabolizable and digestible energy (Exp. 2), and feeding value (Exp. 3) of dry extruded-expelled soybean meal with (DEH) or without (DENH) hulls compared with solvent-extracted soybean meal with hulls removed (SBMNH). Soybeans used to produce DEH were unadulterated prior to extrusion, but those used for DENH were dehulled prior to extrusion. In Exp. 1, six nonlittermate barrows (initially 39 kg) were fitted with ileal T-cannulas and used in a replicated 3 x 3 Latin square design digestion trial. Experimental diets (0.80% total lysine) were cornstarch-based and contained soybean meal from one of the three different sources as the sole source of lysine. Apparent ileal digestibilies of nutrients were similar (P > 0.10) for DEH and DENH. Apparent ileal digestibilies of CP, Lys, Ile, Leu, Arg, Phe, and Val were greater (P < 0.05) for DEH and DENH than for SBMNH. In Exp. 2, six barrows (initially 41 kg) were fed three corn-based diets containing 25% of one of the three soybean meal sources. A fourth diet was fed at the end of the trial containing all ingredients except soybean meal, so that energy values of the soybean meal could be determined by difference. Digestible energy and ME contents were similar (P > 0.10) for DEH and DENH and both had greater (P < 0.05) DE and ME contents than SBMNH. In Exp. 3, pigs (n = 216, initially 10.6 +/- 1.3 kg and 35 +/- 3 d of age) were blocked by weight and allotted to six dietary treatments. Corn-soybean meal-based diets (0.95% digestible lysine and 3.44 kcal/g ME) containing DEH or DENH were compared with similar diets containing SBMNH or solvent-extracted soybean meal with hulls (SBMH). In addition, a diet containing a second expelled soybean meal with hulls (ESBM) was compared with a diet containing SBMH and soy oil. Growth performance of pigs fed diets containing DEH or DENH was not different (P > 0.10) than that of pigs fed corresponding diets containing SMBH or SBMNH. Pigs fed ESBM had lower (P < 0.05) ADG and G/F compared with its corresponding SBMH and soy oil diet. In conclusion, DEH and DENH are more digestible than conventional soybean meal and can be successfully used in swine diets.     

Effects of postweaning dietary energy source on reproductive traits in primiparous sows

An experiment was conducted to study the effects of major dietary energy source fed from weaning to ovulation or from ovulation to d 35 of pregnancy on reproductive traits in primiparous sows. Dietary energy sources were used to manipulate the plasma insulin concentration. One hundred thirteen sows were used in a split-plot design. From weaning to ovulation sows were fed at two times maintenance either a diet with tallow (Fat) or maize starch plus dextrose (Starch) as the major energy source. From ovulation onward, sows within each dietary group were alternately reassigned to either the Fat or the Starch diet and were fed at 1.25 times maintenance. Estrus detection was performed three times a day from d 3 to 9 after weaning and sows were inseminated each day of standing estrus. On d 35 of pregnancy, the sows were slaughtered and their reproductive tracts were removed. Plasma insulin concentration was higher in sows fed the Starch-rich diet than in sows fed the Fat-rich diet on d 4 after weaning (1.30 vs 0.97 ng/mL, P = 0.08) and on d 32 of pregnancy (1.20 vs 0.51 ng/mL, P < 0.001). Plasma glucose and IGF-I concentration on d 4 after weaning and d 32 of pregnancy did not differ between sows on the two dietary energy sources. The percentage of sows exhibiting estrus within 9 d after weaning was 52 and 67% for the Fat and Starch diet before ovulation, respectively (P = 0.11), whereas the weaning-to-estrus interval was 134 vs 123 h, respectively (P = 0.12). Survival analysis showed that sows fed the Fat-rich diet had a 1.6 times higher risk to remain anestrous until d 9 after weaning than sows fed the Starch-rich diet (P = 0.04). No effect of dietary energy source, either before or after ovulation, on uterine, placental, or embryonal development on d 35 of pregnancy was found. It can be concluded that the dietary energy source provided after weaning can affect the risk of sows to remain anestrous but does not affect uterine, placental, or embryonic traits.     

The amino acid profiles of the whole plant and of four plant residues from temperate and tropical forages

This study compared the amino acid (AA) profile of five residues (original forage, borate-phosphate buffer residue (BPR), neutral detergent fiber residue with (NDF+) and without (NDF-) sodium sulfite, and acid detergent fiber residue (ADF). Fourteen grasses and legumes from tropical and temperate regions were used in this study. The use of sodium sulfite did not affect the NDF concentration, but the NDF insoluble protein was lower (P < 0.05) in the NDF+ than in the NDF- (3.9 vs 4.5% DM, respectively). For all of the amino acids tested, the amino acid content, expressed as a percentage of CP, was lower in the ADF residue than in the original forage. There were no differences in the amino acid concentrations of the NDF- and NDF+ extracts (P > 0.05). Only in the case of methionine was there a difference in the amount of amino acid when the original forage was compared with the BPR (1.84 vs 1.45 % CP). When the AA profile of each residue was corrected for the AA content of the ADF, no difference was observed between the AA profile of the original forage and of the BPR (P > 0.05). Similar to the result without correction for the amino acids in ADF, the AA profiles of the NDF+ and NDF- fractions were similar (P > 0.05). From this result, we infer that the sodium sulfite had similar effects on all AA in the NDF residue that we tested. There were differences in amino acid concentrations in the original forage and the NDF residues for several amino acids (Met, Cys, Lys, Thr, Arg, Ile, Leu, and Phe) (P < 0.05). When the amino acid values of the original forage and the BPR were used with animal data in the Cornell Net Carbohydrate and Protein System model, few differences in animal predicted performance were evident. These findings suggest that the AA profile of the original forage can be used to predict the AA profile of the undegraded intake protein instead of using the borate-phosphate buffer residue for amino acid analyses. This would simplify obtaining feed amino acid values for use in the Cornell Net Carbohydrate and Protein System.     

Ergot alkaloid transport across ruminant gastric tissues

Ergot alkaloids cause fescue toxicosis when livestock graze endophyte-infected tall fescue. It is generally accepted that ergovaline is the toxic component of endophyte-infected tall fescue, but there is no direct evidence to support this hypothesis. The objective of this study was to examine relative and potential transport of ergoline and ergopeptine alkaloids across isolated gastric tissues in vitro. Sheep ruminal and omasal tissues were surgically removed and placed in parabiotic chambers. Equimolar concentrations of lysergic acid, lysergol, ergonovine, ergotamine, and ergocryptine were added to a Kreb's Ringer phosphate (KRP) solution on the mucosal side of the tissue. Tissue was incubated in near-physiological conditions for 240 min. Samples were taken from KRP on the serosal side of the chambers at times 0, 30, 60, 120, 180, and 240 min and analyzed for ergot alkaloids by competitive ELISA. The serosal KRP remaining after incubation was freeze-dried and the alkaloid species quantified by HPLC. The area of ruminal and omasal tissues was measured and the potential transportable alkaloids calculated by multiplying the moles of transported alkaloids per square centimeter of each tissue type by the surface area of the tissue. Studies were conducted to compare alkaloid transport in reticular, ruminal, and omasal tissues and to determine whether transport was active or passive. Ruminal tissue had greater ergot alkaloid transport potential than omasal tissue (85 vs 60 mmol) because of a larger surface area. The ruminal posterior dorsal sac had the greatest potential for alkaloid transport, but the other ruminal tissues were not different from one another. Alkaloid transport was less among reticular tissues than among ruminal tissues. Transport of alkaloids seemed to be an active process. The alkaloids with greatest transport potential were lysergic acid and lysergol. Ergopeptine alkaloids tended to pass across omasal tissues in greater quantities than across ruminal tissues, but their transport was minimal compared to lysergic acid and lysergol.     

Fruit and vegetable fiber fermentation by gut microflora from canines

The objective of this study was to assess fermentability by canine gut microflora to include shortchain fatty acid (SCFA) production, organic matter (OM) disappearance, and gas production of vegetable and fruit fiber sources compared to fiber standards (psyllium, citrus pectin, and Solka Floc). Fiber sources included apple pomace, carrot pomace, flaxseed, fruit blend (mixture of peach, almond, nectarine, and plum), grape pomace, pea hulls, pistachio, and tomato pomace. Substrates were fermented in vitro for 4, 12, and 24 h with fecal flora obtained from three healthy dogs. Citrus pectin had the highest OM disappearance, SCFA production, and gas production at all times of fermentation; psyllium was intermediate and Solka Floc was lowest. A wide variation in fermentability was noted among the vegetable and fruit fiber sources. Apple pomace, carrot pomace, and flaxseed had the greatest fermentability as assessed by OM disappearance. Pea hulls and tomato pomace had intermediate OM disappearances, and fruit blend, grape pomace, and pistachio were poorly fermented. Carrot pomace produced the largest amounts of gas and SCFA. Apple pomace produced high concentrations of gas but intermediate concentrations of SCFA. Pea hulls and tomato pomace produced intermediate concentrations of gas and SCFA, whereas flaxseed, fruit blend, grape pomace, and pistachio produced low amounts of these fermentation products. For all substrates collectively, OM disappearance was highly correlated with both gas production (r2 = 0.782 and 0.723 for 12- and 24-h values, respectively) and SCFA production (r2 = 0.737 and 0.738 for 12- and 24-h values, respectively). In general, OM disappearance, gas production, and SCFA production were related to the insoluble:soluble fiber ratio in the samples; as the insoluble:soluble ratio decreased (increased soluble fiber), the OM disappearance, gas production, and SCFA production increased.