Amelia R. Woolums

Live-Cell Characterization and Analysis of a Clinical Isolate of Bovine Respiratory Syncytial Virus, Using Molecular Beacons

ABSTRACT Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 × 103.6 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.

The effect of various Mycoplasma bovis isolates on bovine leukocyte responses

Mycoplasma bovis (M. bovis) contributes to a number of clinical syndromes in cattle; in particular, chronic pneumonia that is poorly responsive to therapy has been increasingly recognized as an important cause of morbidity, mortality, and financial loss. M. bovis impairs host immune function, but little is known about whether field isolates vary significantly in their effect on immune function. This research tested the hypothesis that different field isolates vary in their ability to suppress cellular metabolism and cellular production of radical oxygen species (ROS) by bovine leukocytes. Total blood leukocytes from 6 cattle were exposed to six field isolates, two diagnostic lab isolates, and two high passage laboratory isolates of M. bovis, and ROS production was measured by oxidation of dihydrorhodamine 123 (DHR-123). Cellular metabolism was measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Significant differences in the response to some field isolates were identified. Three field isolates and both diagnostic lab isolates significantly decreased ROS production by leukocytes from multiple cattle, while the high pass laboratory isolates did not. In contrast, MTT reduction was not significantly impaired by any of the M. bovis strains tested. M. bovis impairs ROS production by bovine leukocytes; the magnitude of the effect appears to be isolate-dependent, and is not related to a general impairment of cellular metabolism. Chronic M. bovis infection in some cattle may be related to impaired ability of leukocytes to produce ROS when exposed to M. bovis.

Effect of calf age and administration route of initial multivalent modified-live virus vaccine on humoral and cell-mediated immune responses following subsequent administration of a booster vaccination at weaning in beef calves

OBJECTIVE To compare immune responses following modified-live virus (MLV) vaccination at weaning after intranasal or SC administration of an MLV vaccine to beef calves at 2 or 70 days of age. ANIMALS 184 calves. PROCEDURES Calves were allocated to 1 of 5 groups. The IN2 (n = 37) and IN70 (37) groups received an MLV vaccine containing bovine herpesvirus 1 (BHV1), bovine viral diarrhea virus (BVDV) types 1 and 2, bovine respiratory syncytial virus (BRSV), and parainfluenza 3 virus intranasally and a Mannheimia haemolytica and Pasteurella multocida bacterin SC at median ages of 2 and 70 days, respectively. The SC2 (n = 36) and SC70 (37) groups received a 7-way MLV vaccine containing BHV1, BVDV1, BVDV2, BRSV, parainfluenza 3 virus, M haemolytica, and P multocida SC at median ages of 2 and 70 days, respectively; the control group (37) remained unvaccinated until weaning. All calves received the 7-way MLV vaccine SC at median ages of 217 (weaning) and 231 days. Serum neutralizing antibody (SNA) titers against BHV1, BVDV1, and BRSV and intranasal IgA concentrations were determined at median ages of 2, 70, 140, 217, and 262 days. Cell-mediated immunity (CMI) against BHV1, BRSV, BVDV1, and P multocida was determined for 16 calves/group. RESULTS At median ages of 140 and 217 days, BVDV1 SNA titers were significantly higher for the SC70 group than those for the other groups. Intranasal IgA concentrations and CMI increased over time for all groups. Vaccination at weaning increased SNA titers and CMI in all groups. CONCLUSIONS AND CLINICAL RELEVANCE SC administration of an MLV vaccine to 70-day-old calves significantly increased BVDV1 antibody titers before weaning.

Rapid onset of protection following vaccination of calves with multivalent vaccines containing modified-live or modified-live and killed BHV-1 is associated with virus-specific interferon gamma production

The objective of this study was to determine the effect of vaccination with commercially-available multivalent vaccines containing either modified-live (MLV) bovine herpesvirus-1 (BHV-1) (Bovishield) or MLV plus killed (MLV + K) BHV-1 (Reliant Plus) on protection against challenge at 5 days after a single vaccination. An additional objective was to determine whether cell-mediated immunity as measured by virus-specific interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was associated with any early protection induced by vaccination. Clinical signs, serum neutralizing (SN) titers, and nasal virus isolation (VI) titers were also measured. The 12-16-week-old dairy cross-calves seronegative for antibodies to BHV-1 were vaccinated with a multivalent vaccine containing MLV BHV-1 (n = 19), a multivalent vaccine containing MLV + K BHV-1 (n = 19), or a control multivalent vaccine not containing BHV-1 (n = 10) on day 0 and challenged intranasally on day 5. PBMC were isolated on days 0, 3, 5, 8, 10, 14 and 19. PBMC were incubated in vitro with spent media, live BHV-1, or heat-inactivated BHV-1 for 72 h. Supernatants were assayed for bovine IFN-gamma by ELISA. Bovine herpesvirus-1-specific IFN-gamma production was expressed as percent of the kit positive control, with value for spent media subtracted. Clinical signs were monitored daily. Serum VN titers were measured on days 0-5 and 19. Nasal VI titer was measured every other day from days 5 to 19. Interferon gamma production was higher on day 5, and was significantly increased post-challenge, in both vaccine groups compared to controls. There was no difference between vaccine groups on any day. There was no significant difference in SN titer among groups on any day. Virus isolation titer was significantly higher in controls on days 6 and 8 compared to both vaccine groups. Temperatures were significantly higher and nasal discharge was present more often post-challenge in controls compared to vaccine groups. Vaccination 5 days prior to challenge with commercially-available vaccine containing MLV or MLV + K BHV-1 was associated with increased BHV-1-specific IFN-gamma production, decreased viral shedding, lower temperatures and less nasal discharge post-challenge. Cell mediated immune responses as measured by IFN-gamma production are stimulated rapidly following BHV-1 vaccination of calves.

Pathogenesis of a Bovine Enterovirus-1 Isolate in Experimentally Infected Calves

The pathogenesis and virulence of Bovine enterovirus-1 (BEV-1) in cattle is largely unknown. Reports concerning its virulence suggest that there might be an association between BEV-1 infections and a range of diseases in cattle that vary from respiratory to enteric to reproductive disease and infertility. In the current study, the pathogenesis associated with acute infection of BEV-1 in calves experimentally inoculated with the Oklahoma isolate of BEV-1 was described. Although interpretation of the study was limited by lack of an effective control group, results suggest that an association between inoculation of BEV-1, virus localization, and the potential development of lesions in the brain and heart probably exists. In the experiment, BEV-1 virus localized to the terminal ileum, ileocecal and cecocolonic junctions, spiral colon, and ileocecal lymph nodes; BEV-1 virus was detected in the cytoplasm of enterocytes, lamina propria macrophages, endothelium, neurons of the submucosal and myenteric plexi, and lymphocytes of the submucosal lymphoid tissue. Although no clinical signs were noted following acute infection, BEV-1 was localized in the cerebellar white matter of a calf with encephalitis and in the heart of another calf with coronary arteritis. The current study suggests that the BEV-1 isolate is infectious to young calves and that BEV-1 potentially can have a similar pathogenesis to that observed in natural or experimental enterovirus infections in other species.

Producer survey of herd-level risk factors for nursing beef calf respiratory disease

OBJECTIVE To identify herd-level risk factors for bovine respiratory disease (BRD) in nursing beef calves. DESIGN Population-based cross-sectional survey. SAMPLE 2,600 US cow-calf producers in 3 Eastern and 3 Plains states. PROCEDURES The associations of herd characteristics with BRD detection in calves and cumulative BRD treatment incidence were determined. RESULTS 459 (177%) surveys were returned and met the inclusion criteria; 48% and 52% of these surveys were completed by producers in Plains and Eastern states, respectively. Mean (95% confidence interval) number of animals in herds in Plains and Eastern states were 102 (77 to 126) and 48 (40 to 56), respectively. Bovine respiratory disease had been detected in ≥ 1 calf in 21% of operations; ≥ 1 calf was treated for BRD and ≥ 1 calf died because of BRD in 89.2% and 46.4% of operations in which calf BRD was detected, respectively. Detection of BRD in calves was significantly associated with large herd size, detection of BRD in cows, and diarrhea in calves. Calving season length was associated with BRD in calves in Plains states but not Eastern states. Cumulative incidence of BRD treatment was negatively associated with large herd size and examination of cows to detect pregnancy and positively associated with calving during the winter, introduction of calves from an outside source, offering supplemental feed to calves, and use of an estrous cycle synchronization program for cows. CONCLUSIONS AND CLINICAL RELEVANCE Results of this study indicated factors associated with calf BRD risk; modification of these factors could potentially decrease the incidence of BRD in nursing calves.

Comparative efficacy of enrofloxacin and tulathromycin for treatment of preweaning respiratory disease in dairy heifers

Preweaning respiratory disease continues to have a substantial effect on the current and future productivity of dairy replacement animals. Establishing an effective treatment plan for the preweaned calf may have a significant effect on well-being and lifetime productivity by limiting any early development of chronic disease. The primary objective of this study was to examine the efficacy of treatment with tulathromycin (TUL) or enrofloxacin (ENR) on the risk of re-treatment, with a secondary objective of investigating the effect of disease and subsequent treatment choice on average daily gain (ADG). A total of 1,141 Holstein heifers from 4 farms were observed and systematically scored for evidence of respiratory disease from birth through weaning or the time of death. At the time of diagnosis, calves were randomly and blindly allocated into 2 treatment groups. The overall incidence of respiratory disease was 60.9%. In the univariable analysis, the incidence of re-treatment between 7 and 10d of initial therapy for calves treated with ENR was greater than that in calves treated with TUL (27.6 vs. 21.2%). After adjusting for farm ID, clinical score at first treatment, and weight at first treatment, the odds of re-treatment were 1.5 times higher for calves treated with ENR than with TUL. The percentage of calves that required more than one re-treatment was higher for calves that received ENR compared with those that received TUL (9.3 vs. 4.1%). We observed no difference in ADG between calves treated with ENR or TUL, and no difference in ADG between calves that were treated for respiratory disease and those that were not treated for respiratory disease. Appropriate drug therapy for preweaning respiratory disease may have an important role in reducing the odds of re-treatment during the preweaning period.

Agreement Among 4 Sampling Methods to Identify Respiratory Pathogens in Dairy Calves with Acute Bovine Respiratory Disease

BACKGROUND Four sampling techniques commonly are used for antemortem identification of pathogens from cattle with bovine respiratory disease (BRD): the nasal swab (NS), guarded nasopharyngeal swab (NPS), bronchoalveolar lavage (BAL), and transtracheal wash (TTW). Agreement among these methods has not been well characterized. OBJECTIVE To evaluate agreement among TTW and NS, NPS, or BAL for identification of viral and bacterial pathogens in dairy calves with BRD. ANIMALS One hundred dairy calves with naturally acquired BRD. METHODS Calves were sampled by all 4 methods. Viral agents were identified by real-time RT-PCR, bacteria were identified by aerobic culture, and Mycoplasma bovis (M. bovis) isolates were speciated by PCR. Agreement among TTW and NS, NPS, or BAL was evaluated by calculating the kappa statistic and percent positive agreement. McNemars exact test was used to compare the proportions of positive results. RESULTS Agreement among TTW and NS, TTW and NPS, and TTW and BAL, was very good for identification of P. multocida, M. haemolytica, and M. bovis. For bovine respiratory syncytial virus (BRSV), agreement with TTW was moderate for NS, good for NPS, and very good for BAL. For bovine coronavirus (BCV), agreement with TTW was moderate for NS and NPS, and good for BAL. McNemars test was significant only for BCV, indicating that for this pathogen the proportion of positive results from NS and NPS could not be considered comparable to TTW. CONCLUSIONS AND CLINICAL IMPORTANCE This study provides guidance for veterinarians selecting diagnostic tests for antemortem identification of pathogens associated with BRD.

Prevalence of bacteremia in dairy cattle with acute puerperal metritis.

Background Acute puerperal metritis (APM) affects 30% of postpartum dairy cattle. Bacteremia negatively impacts survival in cattle with coliform mastitis. However, the prevalence of bacteremia in dairy cattle with APM is unknown. Hypothesis Bacteremia is detectable in a large proportion of cattle with APM. Animals Seventeen dairy cows with APM and 17 healthy dairy cattle. Methods Prospective case‐control study. Cases were identified by daily monitoring of cattle in the first 10 days after calving. Controls were matched to cases by parity and days in milk. Cows were examined at the time of identification of APM. A complete blood count, serum biochemical analysis, and bacteriologic culture of blood and lochial fluid were performed on each animal at the time of diagnosis. The same samples were collected from healthy herdmates of a similar parity and days in milk. Blood culture results and clinicopathologic variables were compared between groups. Conditional logistic regression was used to evaluate factors associated with APM, whereas multivariate logistic regression was used to evaluate factors associated with bacteremia. Results Bacteremia occurred in 53% (9/17) of cattle with APM and 53% (8/15) controls. Bacillus spp. was the organism most commonly isolated from the bloodstream in cattle of both groups. Bacteremic cattle in both groups were significantly less likely to have basophils in the peripheral circulation (P = .02) and more likely to have higher serum globulin concentrations (P = .02). Conclusions and Clinical Importance Bacteremia is a common occurrence in postpartum dairy cattle. Further study is warranted to investigate the modes by which bacteria colonize the bloodstream in this population of animals and the importance of bacteremia on health and productivity of affected animals.

Analysis of mRNA expression for genes associated with regulatory T lymphocytes (CD25, FoxP3, CTLA4, and IDO) after experimental infection with bovine viral diarrhea virus of low or high virulence in beef calves

Abstract Immunosuppression caused by bovine viral diarrhea virus (BVDV) has been associated with lymphocyte depletion, leukopenia and impairment of leukocyte function; however, no work has been done on the relationship between BVDV and regulatory T lymphocytes (Tregs). The objective of this study was to compare the mRNA expression of genes associated with Tregs (CD25, FoxP3, CTLA4, and IDO), after experimental infection of beef calves with low (LV) or high (HV) virulence BVDV. Thirty BVDV-naïve calves were randomly assigned to three groups. Calves were intra-nasally inoculated with LV (n =10, strain SD-1) or HV (n =10, strain 1373) BVDV or BVDV-free cell culture medium (control, n =10). Quantitative RT-PCR was used to determine the expression of target genes in tracheo-bronchial lymph nodes and spleen on day 5 post-infection. The mRNA expression of CD25 was up-regulated in tracheo-bronchial lymph nodes of LV (P <0.05), but not in HV compared to the control group. The expression of FoxP3 and CTLA4 was not increased in tracheo-bronchial lymph nodes of either of the BVDV-inoculated groups. A dramatic up-regulation of IDO mRNA was observed in tracheo-bronchial lymph nodes of LV (P <0.05), but not HV compared to the control calves. In conclusion, experimental infection with BVDV did not provide evidence of Treg activation based on expression of FoxP3 and CTL4. Differential expression of CD25 and IDO mRNA on day 5 post-infection with HV or LV BVDV might reflect temporal differences in transcription occurring during the immune response elicited by these viral strains, or differences in viral infectivity of the host cells.