Risk factors for seroprevalence of ovine lentivirus in breeding ewe flocks in Nebraska, USA.

The prevalence of and risk factors for ovine lentivirus (OLV) infection in 1466 breeding ewes in nine US Meat Animal Research Center (MARC) flocks were determined using a recombinant transmembrane protein (PTM) enzyme-linked immunosorbent assay (ELISA) to detect serum anti-OLV antibodies and define infection. Based on multivariable logistic regression, confinement birth and rearing (odds ratio (OR) = 1.6), older weaning ages (OR = 1.1 week-1), and older age (OR = 1.3-2.5 year-1 beyond age 1 year) were significantly associated with higher OLV prevalence in ewes. Prevalence also varied significantly by flock, with Finnsheep and Texel ewes having the highest prevalences and Booroola Merino and Suffolk ewes having the lowest prevalences. These findings support the hypothesis that management control efforts should concentrate on events early in the life of sheep, as this period is associated with factors which can modulate the risk for OLV infection.

Effect of ewe ovine lentivirus infection on ewe and lamb productivity.

We used a previously described sensitive and specific ovine lentivirus (OLV) recombinant transmembrane (rTM) protein enzyme-linked immunosorbent assay (ELISA) to detect anti-OLV antibodies and define OLV infection in breeding ewes from nine US Meat Animal Research Center (MARC) flocks. We estimated the production impacts of dam rTM ELISA seropositivity on ewe and lamb productivity in the birth-to-weaning interval using production data from 1466 breeding ewes (of which 1242 actually lambed) and their 2452 lambs born in spring 1992 using several multiple linear and logistic regression models. By adjusting for lamb weaning age, gender, type of birth and rearing, birth difficulty, dam age, and flock, the component of ewe or lamb productivity related to ewe OLV infection alone was isolated. The rTM ELISA-negative ewes produced significantly more total weight of weaned lamb per ewe-lambing (3.84 kg) and per ewe ram-exposed (4.95 kg) compared to their OLV-positive flockmates. Negative ewes also weaned 0.11 more lambs per ewe-lambing and 0.09 more lambs per ewe ram-exposed, gave birth to 0.13 more lambs per ewe ram-exposed, and were more likely to lamb after breeding (odds ratio (OR) = 1.9) compared to equivalent OLV-positive ewes. Lambs reared by OLV-negative ewes weighed 0.15 kg more at birth, gained 8 g more per day through weaning, and weighed 0.59 kg more at 56-day weaning. Preweaning mortality was lower (OR = 0.8) among lambs born to OLV-negative compared to OLV-positive ewes, although this difference was not significant. Our results suggest that subclinical OLV infection has important detrimental effects on sheep production which occur in cumulative fashion from breeding through weaning and that OLV control efforts may be financially justified in some sheep flocks.

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157.

Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) may react with several other bacteria including Brucella abortus, Brucella melitensis, Yersinia enterocolitica O9, Escherichia hermannii, and Stenotrophomonas maltophilia. We produced eight monoclonal antibodies (MAbs) specific for the LPS of E. coli O157. Western blots (immunoblots) of both the phenol phase (smooth) and the aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivities to Salmonella O30 LPS (group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157 tested, which included 47 isolates of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of non-E. coli gram-negative enteric bacteria.

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.

Relationships among treatment for respiratory tract disease, pulmonary lesions evident at slaughter, and rate of weight gain in feedlot cattle.

OBJECTIVE: To quantify the effects of treatment for clinical respiratory tract disease and pulmonary lesions identified at slaughter on rate of weight gain in feedlot cattle. DESIGN: Prospective longitudinal study. ANIMALS: 469 feedlot steers. PROCEDURE: Clinical respiratory tract disease was monitored between birth and slaughter. Steers were weaned at approximately 6 months old and entered into the feedlot for a mean of 273 days. Mean daily weight gain (MDG) was monitored during the feeding period. Lungs were collected at slaughter and evaluated for gross lesions indicative of active or resolved pneumonia. RESULTS: Mean daily weight gain during the feeding period was 1.30 kg, and ranged from 1.16 to 1.46 kg within individual pens. Thirty-five percent of steers received treatment for respiratory tract disease between birth and slaughter, whereas 72% had pulmonary lesions evident at slaughter. Among steers treated for clinical respiratory tract disease, 78% had pulmonary lesions, whereas 68% of untreated steers had pulmonary lesions. Pulmonary lesions at slaughter were associated (P < 0.01) with a 0.076-kg reduction in MDG during the feeding period. Treatment for clinical disease was not associated with MDG after adjustment for the effect of pulmonary lesions. CLINICAL IMPLICATIONS: Treatment of clinically affected feedlot cattle may be inadequate to prevent significant production losses attributable to respiratory tract disease.

Ovine lentivirus antibody detection in serum, colostrum and milk using a recombinant transmembrane protein ELISA.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against ovine lentivirus (OLV) in serum, colostrum, and milk from naturally infected sheep. The assay used OLV recombinant transmembrane envelope protein (rTM) as a test antigen. Matched serum/colostrum and serum/milk samples were collected at 24h, 4 weeks (mid-lactation), and 8 weeks (weaning) post-lambing. Among 129 paired samples collected at 24 h post-lambing, there was overall test agreement (concordance) of 82.9% and a kappa value of 0.658 between serum and colostrum rTM ELISA results. Among 130 mid-lactation samples, the milk ELISA had 100% specificity and 64.9% sensitivity relative to the serum ELISA, there was concordance of 79.2%, and a kappa value of 0.602. At mid-lactation, the serum agar gel immunodiffusion test had a sensitivity of 0.390 and 0.560 relative to the serum and milk rTM ELISAs, respectively. Matched serum and milk rTM ELISA results at weaning were very similar to those at mid-lactation. Finally, increased occurrence and severity of subclinical mastitis at weaning was found in ELISA-seropositive compared with ELISA-seronegative ewes. Both subclinical mastitis and ewe OLV infection had a negative impact on lamb growth and weaning weights. Compared with blood, colostrum and milk are easier and less expensive to sample and store. These results suggest that rTM ELISA testing of colostrum and milk could be used to supplement serologic testing in OLV screening or eradication programs.

Haptoglobin response to clinical respiratory tract disease in feedlot cattle.

OBJECTIVE: To quantify haptoglobin response to respiratory tract disease in feedlot cattle, and to investigate its ability to predict disease outcome and response to antibiotic treatment. DESIGN: Randomized clinical trial. ANIMALS: 60 feedlot calves with clinical respiratory tract disease. PROCEDURE: Calves were randomly assigned to receive a standard antibiotic treatment regimen (TRT), or to observation pens without antibiotic treatment. Serum haptoglobin concentration was measured at initial and final examinations. Calves were examined for presence of gross pulmonary lesions at slaughter. RESULTS: Mean +/- SD serum haptoglobin concentration at initial examination was 67 +/- 108 mg/dl, with range of 0 to 508 mg/dl. Haptoglobin concentration at initial examination was similar for the TRT group and the group that did not receive antibiotic treatment, but at final examination, TRT-group calves had lower (P < 0.01) mean values. Calves receiving antibiotic treatment had haptoglobin concentration at or near zero at final examination. Calves not receiving antibiotic treatment had only slightly lower mean haptoglobin concentration at final examination, compared with initial examination. Within treatment groups, haptoglobin concentration was similar for cases with different outcomes. Calves with gross pulmonary lesions at slaughter had numerically higher, although statistically similar, haptoglobin concentrations at initial examination, compared with calves without lesions. CONCLUSIONS: Feedlot cattle with clinical respiratory tract disease have a large and variable haptoglobin response. Antibiotic treatment resulted in lower serum haptoglobin values, although low values were not required for full clinical recovery. CLINICAL RELEVANCE: Serum haptoglobin concentration may be an indicator of response to antibiotic therapy, although it appears to be unrelated to case severity or need for treatment.

Serum haptoglobin concentrations in a population of feedlot cattle.

OBJECTIVE: To determine serum haptoglobin concentrations in a population of feedlot cattle and evaluate their usefulness in predicting subsequent clinical respiratory tract disease. DESIGN: Prospective longitudinal study. ANIMALS: 366 beef calves. PROCEDURE: Serum samples were obtained at feedlot entry and 40 and 65 days on feed (DOF). Calves were observed daily for clinical signs of respiratory tract disease. The lungs of 144 of the calves were evaluated at slaughter for the presence of gross lesions of pneumonia. RESULTS: 58% of the calves had detectable serum haptoglobin concentration in at least 1 sample. The proportion of calves with detectable haptoglobin were similar at each sample collection time. A higher proportion of the calves had values > 10 mg/dl at 40 DOF. The proportion of calves observed with clinical disease during the 10-day period after the 40 DOF sample increased (P < 0.10) as serum haptoglobin concentration increased. At 65 DOF, calves with serum haptoglobin value > 10 mg/dl had a higher (P < 0.05) rate of subsequent clinical respiratory tract disease than did calves with lower values. The proportion of calves with gross pulmonary lesions slaughter increased (P < 0.05) from 39% among calves without detectable serum haptoglobin concentration in any of the 3 samples to 63% among calves with at least 1 observed value > 10 mg/dl. CONCLUSIONS: We observed associations between serum haptoglobin concentration and subsequent clinical respiratory tract disease and pulmonary lesions at slaughter. However, serum haptoglobin concentration alone is not adequate for prediction of clinical disease. CLINICAL RELEVANCE: The usefulness for cross-sectional sampling of serum haptoglobin concentration as a diagnostic tool for clinical respiratory tract disease in feedlot cattle appears to be limited.

Use of the polymerase chain reaction for Salmonella detection.

A primer set of oligonucleotides (S18 and S19) from the ompC gene of Salmonella has been evaluated for specific detection of Salmonella by polymerase chain reaction (PCR). This primer set successfully amplified 40 Salmonella serovars (60 isolates), but not 24 non-Salmonella bacteria (42 isolates) that have been tested so far. The uniqueness of these primer sequences was also confirmed. The sensitivity of PCR detection in extracted chromosomal DNA for Salm. typhimurium was 1 pg. The sensitivity for boiled whole bacteria was 400 cells. The detection of Salm. typhimurium in ground beef samples required 4-6 h enrichment with an initial inocula of 100 bacteria.

The heterogeneity of bovine IgG2. VII. The phenotypic distribution of the A1 and A2 allotypes of IgG2a among beef cows with known clinical history.

A1 and A2 are allotypes of bovine IgG2a which differ significantly in their primary structure, allotope expression and the products of pepsin digestion. An analysis of 754 beef cows from 14 different breeds at the Meat Animal Research Center (MARC), Clay Center, NE, demonstrated a significant difference in the distribution of A1 and A2 among breeds but failed to find any correlation between the clinical disease history of the animals tested and their A-allotype. The proportion of all animals with either a history of infectious or respiratory disease (43.3 +/- 3.5 and 17 +/- 0, respectively) was the same among A1/A1, A1/A2 and A2/A2 animals. Similarly, there was no preferential association between allotype and clinical disease within any one breed. A very high incidence of A1 homozygotes was found among Angus (84%), Brown Swiss (100%), Limousin (87%), MARC I (87%) and Pinzgauers (88%). In contrast, Herefords had a high incidence of A2/A2 homozygotes (41%) as did Brahmans (46%) and Gelbveih (34%). The distribution of A1/A1, A1/A2 and A2/A2 animals within any breed was totally consistent with the concept that A1 and A2 represent Mendelian co-dominant alleles. These data suggest that, among vaccinated female beef cattle in a normal environment, A-allotypy plays no role in the propensity for clinical disease as defined in this study. It does not rule out such an association in non-vaccinated, severely stressed animals and in calves exposed to severe outbreaks of an infectious agent.

Production and identification of recombinant proteins of Salmonella typhimurium and their use in detection of antibodies in experimentally challenged animals.

Antibodies to experimental Salmonella typhimurium challenge in cattle and sheep were assessed by 15 recombinant flagellum proteins. The 15 DNA fragments selected for gene expression were derived from external flagellin, hook, hook-associated protein, and basal body gene domains. Our efforts were focused on characterizing the humoral immune response of Salmonella infected and vaccinated animals and identifying immunodominant antigenic determinants. This communication reports that the 159-261 amino acids of external flagellum (FliCi-1), 285-331 amino acids of hook protein (FlgE-2), and 309-391 amino acids and 440-537 amino acids of hook-associated protein (FlgK-1 and -2) appeared to be the most immunoreactive proteins and were recognized by all of the experimental animal sera tested in this study.

Effect of breed, intake, and carcass composition on the status of several macro and trace minerals of adult beef cattle.

The objective was to determine the association between breed, intake, and carcass composition and the status of liver Cu, Zn, and Fe, and serum Cu, Zn, Ca, and Mg of 118 mature cows representing nine breeds of cattle. Breeds used were Angus, Braunvieh, Charolais, Gelbvieh, Hereford, Limousin, Red Poll, Pinzgauer, and Simmental. The cows were fed one of four levels of DMI: 58, 76, 93, and 111 g of DMI.wt-75.d-1. A ground alfalfa, corn, and corn silage diet was fed for up to 5 yr. There was no relationship between liver and serum concentrations of Cu, a negative correlation (P < .05) existed between liver and serum concentrations of Zn and a positive correlation (P < .01) existed between liver concentrations of Cu and Zn. Concentrations of serum Ca were positively correlated (Cu and Zn, P < .01; Mg, P < .05) with serum concentrations of Cu, Zn, and Mg, but negatively correlated (P < .01) with liver Fe. Liver Cu was higher (P < .05) for the Limousin breed than all others, except Angus. Liver Zn concentrations were higher (P < .05) for Limousin than for Pinzgauer, but no other breed differences were observed. Liver Cu concentration was not affected by daily intake, but liver Zn concentration increased (P < .05) with increased daily intake. Liver Fe concentration decreased (P < .01) in a curvilinear manner with increased daily intake. No breed differences in serum concentrations of Cu or Zn were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Variation of abscess formation in cattle after vaccination with a modified-live Pasteurella haemolytica vaccine.

During the spring of the first year of a vaccine study, 57 of 238 calves (24%), in which modified-live Pasteurella haemolytica vaccine (MLV) was injected twice, developed 1 or more abscesses. Abscesses were not observed after multiple visual examinations of 437 calves given killed P haemolytica bacterin or placebo injections of similar adjuvants used in the vaccine and bacterin. Calves that developed abscesses after the second injection of MLV weighed significantly (P < 0.05) less (on the basis of body weight adjusted for weaning weight) at the second injection than did those that did not develop abscesses. Compared with calves given MLV that did not develop observable abscesses, calves developing abscesses after the second injection of MLV weighed 11.0 and 14.2 kg less, respectively, at 56 days and 112 days after injection, and they had 11.0 kg less gain at 56 days after injection. Abscess prevalence tended to be highest on certain days or at certain locations used for cattle processing, and the prevalence of abscesses increased in cattle processed later on a given day. Abscesses were not observed in 2 other groups of similarly treated calves vaccinated in the autumn or in the subsequent spring.

Evaluation of an ELISA for detection of ovine progressive pneumonia antibodies using a recombinant transmembrane envelope protein.

An enzyme-linked immunosorbent assay (ELISA) was developed using a recombinant protein corresponding to the N'-terminal hydrophilic region of transmembrane glycoprotein (TM) of ovine lentivirus. This assay reproducibly detected antibodies in sera from 207 of 212 ovine progressive pneumonia (OPP) virus-infected sheep, and the recombinant TM ELISA accurately identified 26% (35 vs. 9) more seropositive samples than did the agar gel immunodiffusion test when applied to 100 sera from an infected flock. This assay also yielded no false-positive results in 14 true negative sera. Results of these experiments were further confirmed by the recombinant TM and recombinant p25 Western blot assay. A single recombinant TM antigen, as the coating antigen in ELISA, can be used successfully for the detection of OPP virus-infected animals and can improve the sensitivity and specificity for OPP diagnosis.

Effect of sire and dam breed on copper status of fat lambs.

To determine the effect of breed on copper status of crossbred (F1) lambs, a study was conducted using 187 crossbred wether lambs. Wether lambs were progeny of five ram breeds (Dorset, Finnsheep, Montadale, Romanov, and Texel) and two ewe breeds (Rambouillet and a composite breed [1/2 Columbia, 1/4 Suffolk, 1/4 Hampshire]). Diets were adjusted to decrease crude protein twice during the experimental period, with analyzed copper levels for the three diets of 5.2, 4.4, and 3.6 ppm. All means were adjusted to a constant carcass weight of 27.265 kg using a covariate analysis. Least squares means of the liver copper concentration for sire breeds ranged from 307 to 458 micrograms/g on a DM basis. Romanov- (307 micrograms/g) and Finnsheep-sired (327 micrograms/g) lambs had the lowest liver copper concentration, whereas Montadale- (359 micrograms/g) and Dorset-sired (360 micrograms/g) lambs were intermediate and Texel-sired (458 micrograms/g) lambs were significantly higher compared with other lamb breeds. Least squares means for total liver copper for the five ram breeds ranged from 69 to 101 mg. Finnsheep- (69 mg), Romanov- (72 mg), and Montadale-sired (80 mg) lambs were lowest, Dorset-sired (85 mg) lambs were intermediate, and Texel-sired (101 mg) lambs were higher (P < .02) compared with other lamb breeds for total liver copper. Least squares means for serum copper ranged from .86 to 1.05 ppm. Levels of serum copper were similar for Montadale- (.86 ppm), Finnsheep- (.90 ppm), Texel- (.93 ppm), and Dorset-sired (.94 ppm) lambs and higher (P < .02) for Romanov-sired (1.05 ppm) lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant polypeptide from the gp48 region of the bovine viral diarrhea virus (BVDV) detects serum antibodies in vaccinated and infected cattle.

To characterize the immune response of cattle to bovine viral diarrhea virus (BVDV) glycoprotein gp48, we have produced a large amount of recombinant glutathione-s-transferase-gp48 (GST-gp48) fusion protein in Escherichia coli. Antibodies to gp48 were present in cattle vaccinated with killed or modified-live virus vaccination, or following natural infection. These results were in agreement with results of serum neutralization (SN) test which detected gp53 of BVDV.

Efficiency of various cloned DNA probes for detection of bovine viral diarrhea viruses.

We have evaluated 24 cytopathic (CP) and 37 noncytopathic (NCP) strains of bovine viral diarrhea virus (BVDV) with a dot blot assay using four different genome segments of the NADL strain as hybridization probes (p80, p54, gp53, and gp62). The p80 and p54 probes hybridized to 23/24 (96%) and 22/24 (92%), respectively, of CP strains examined. In contrast, these same two probes only detected 16/37 (43%) and 5/37 (13%), respectively, of the NCP strains examined. The gp53 probe detected 18/24 (75%) and the gp62 probe detected 19/24 (79%) of the CP strains. In contrast, these latter two probes only detected 9/37 (24%) and 7/37 (20%), respectively, of NCP strains. This low detection rate of NCP strains suggests a need for developing a probe based on NCP sequences for identification of NCP strains.

Expression of the p80 region of bovine viral diarrhea virus and identification of specific antibodies to this recombinant protein in bovine sera.

A 917-base pair segment of the bovine viral diarrhea virus (BVDV) genome encoding part of the p80 region was cloned into plasmid Gex-2T expression vector for expression as a fusion protein with glutathione-S-transferase (GST). When the p80 and GST sequences were in the same reading frames, the resulting GST-p80 fusion protein had a molecular mass of 58 kilodaltons (kDa) in SDS-PAGE. Extracts of control E. coli carrying only the vector plasmid (Gex-2T) did not contain this new 58-kDa protein band. Mouse monoclonal antibody specific to BVDV-p80 recognized this recombinant protein. Seventy cattle sera that had an SN titer (to TGAC isolate of cytopathic BVDV) greater than 1:8 reacted with this recombinant protein in Western blots. Of 28 cattle sera that had SN titers less than or equal to 1:8, only one serum tested positive on Western blots.

Serologic detection and practical consequences of antigenic diversity among bovine viral diarrhea viruses in a vaccinated herd.

Samples of sera were obtained from 5,725 cows in a semiclosed herd. In each of the preceding 7 years, the herd was vaccinated against bovine viral diarrhea (BVD) with killed virus. Neutralizing antibody tests were done on all samples of sera, using cytopathic virus, BVD-TGAC virus, that was antigenically distinct from the vaccine virus. Most samples of sera had high titers of neutralizing antibodies against BVD-TGAC virus. In 48 samples of sera, neutralizing antibodies were not detected against BVD-TGAC virus, but were detected against the vaccine virus. Neutralizing antibodies against selected noncytopathic BVD viruses were not detected in several samples of serum that had neutralizing antibodies against the vaccine virus and BVD-TGAC virus. Noncytopathic BVD virus was isolated from sera obtained from 3 cows less than 4 years old. Two cows were available for further testing, and persistent infection with BVD virus was confirmed in both cows. The BVD viruses isolated from those cows were not neutralized by several samples of sera. Immunoprecipitation of polypeptides induced by the vaccine virus was done with selected samples of serum. Two patterns of immuno-precipitated viral-induced polypeptides were identified. One pattern was consistent with exposure of cows with live virus. The other pattern was consistent with exposure of cows with only the killed virus vaccine.