Evaluation of cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, and vesicular stomatitis virus in vitro

OBJECTIVE: To evaluate cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against cytopathic and noncytopathic bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), and vesicular stomatitis virus (VSV) in vitro. SAMPLE POPULATION: Primary bovine testicular cells and Mardin Darby bovine kidney cells. PROCEDURES: To evaluate cytotoxicity, cells were added to serial dilutions of each interferon. To evaluate antiviral activity of each interferon, interferons were serially diluted 1:10, and tissue culture cells were added; virus was then added at 3 time points. Prevention of viral infection by interferon was defined as failure to induce cytopathologic effect for VSV, IBRV, and cytopathic BVDV and failure to detect virus immunohistochemically for cytopathic and noncytopathic BVDV. RESULTS: No evidence of cytotoxicity in either cell line was detected after incubation with interferon alfa-2a or interferon alfa-B/D. However, reduced growth rates of tissue culture cells were detected for each interferon when undiluted interferon was tested. Comparable and profound antiviral activities against cytopathic and noncytopathic BVDV were evident for each interferon. Interferon alfa-2a and interferon a-B/D had comparable antiviral activities against VSV. Neither interferon had antiviral activity against IBRV. CONCLUSIONS AND CLINICAL RELEVANCE: The safety and marked in vitro antiviral activity against noncytopathic BVDV, cytopathic BVDV, and VSV suggest that interferons alfa-2a and alfa-B/D may be useful for treatment of natural disease after infection with these viruses.     

Leukocyte profile of cattle persistently infected with bovine viral diarrhea virus

Animals acutely infected with bovine viral diarrhea virus (BVDV) exhibit transient immunosuppression as a result of the virus' predilection for cells that play critical roles in the host immune system. Acute BVDV infections have major effects on thymic and follicular T-lymphocytes, as well as follicular B-lymphocytes, often resulting in severe reduction in circulating numbers of lymphocytes and suppression of functional activities of these cells. Granulocytes and monocytes are equally susceptible to BVDV infections with reduction in numbers and suppression functions. However, there is limited information on the leukocyte profile of cattle persistently infected (PI) with BVDV. This study reports on phagocytic activities of granulocytes and monocytes as well as immunophenotyping by flow cytometric analysis of leukocytes isolated from healthy non-PI (NPI) and PI animals. No significant differences were found between the leukocyte profiles and the phagocytic activities of PI animals when compared to a group of healthy NPI animals.     

Total protein and immunoglobulins G1, G2, and M in serum of calves persistently infected with bovine viral diarrhea virus

Total serum protein and immunoglobulins (Ig) G1, G2, and M concentrations were investigated in 11 calves persistently infected with bovine viral diarrhea virus. These calves were allowed to suckle from their dams until weaned. A gradual increase in total protein was observed from birth to 12 months of age. There was a wide variation in Ig concentrations in pre- and postcolostrum sera. The IgG1 increased from the time of delivery of the calves to the 5th month, decreased by the 10th month, and then stabilized through the 12th month. The IgG2 increased from birth to 10 months and remained stable through 12 months. The IgM increased from birth to the 11th month, and then decreased sharply by the 12th month.     

Identification of herds with cattle persistently infected with bovine viral diarrhea virus by virological evaluation of three calves

For the identification of herds with cattle persistently infected (PI) with bovine viral diarrhea virus, 1,272 animals from 20 herds were subjected to serum neutralizing (SN) test using the Nose strain and virus isolation. Eighteen PI cattle were detected from 5 herds. On the phylogenetic tree based on the nucleotide sequences of the 5' untranslated region, the isolates from the PI cattle were classified into genotypes-1a or -1b. Of 3 unvaccinated calves aged 6 to 12 months selected from each herd, the probabilities of obtaining 2 or more non-PI cattle with SN antibody titers of 64 or more (P(SN)), one or more PI cattle (P(VI)), and either of the conditions (P(Total)) were calculated using the hypergeometric probability model. P(Total) for the 5 herds with PI cattle was 1.000. P(SN) for 3 herds with many PI cattle within the selected age group was as low as 0.500 or less, and P(VI) was as high as 0.886 or more. P(SN) in the 2 other herds with few PI cattle was 1.000, and P(VI) was as low as 0.375 or less. P(Total) in 13 of 15 herds without PI cattle was 0.000, and was 0.714 or 0.774 for the 2 other herds. These results suggest that herds with PI cattle can be predicted with high accuracy when both SN test and virus isolation are performed on only 3 unvaccinated calves aged 6 to 12 months selected from a herd.     

Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus

The relationship between bovine viral diarrhea virus (BVDV) infection and thrombocytopenia was studied in 18 veal calves experimentally infected with BVDV. All calves were free of BVDV, and 13 calves were free of serum neutralizing antibodies to BVDV before virus inoculation. Calves were inoculated at approximately 10 days of age, and platelet counts were monitored over a period of several weeks. Ten additional calves housed in close proximity were kept as uninoculated controls. A profound decrease in platelet counts by 3 to 11 days after inoculation was seen in all calves that had neutralizing antibody titers less than 1:32 before infection. Severe thrombocytopenia (less than 5,000 platelets/microliter) was seen in 12 calves, 11 of which also developed hemorrhages. Necropsy findings in 3 severely thrombocytopenic calves that died included multiple hemorrhages throughout the body. Calves that recovered had increased platelet counts, and in most instances, a corresponding increase in neutralizing antibody titers to BVDV. At 11 days after inoculation, BVDV was detected on platelets by use of immunofluorescence, but evidence of surface-bound immunoglobulin was not found. The results suggest that a nonimmunoglobulin-mediated method of platelet destruction or sequestration develops as a sequela to BVDV infection.     

Prevalence of calves persistently infected with bovine viral diarrhea virus in beef cow-calf herds enrolled in a voluntary screening project

OBJECTIVE: To report the prevalence of bovine viral diarrhea virus (BVDV) in calves and calf groups (ie, calves from the same farm) in beef breeding herds and evaluate the ability of biosecurity risk assessment questionnaires to identify calf groups with positive results for BVDV. DESIGN: Nonrandom survey. ANIMALS: 12,030 calves born in spring from 102 operations. PROCEDURES: Cow-calf producers that voluntarily enrolled in a screening project submitted ear notch specimens from calves and answered a 29-question survey instrument. Ear notch specimens were tested for BVDV with an antigen-capture ELISA (ACE), and ear notch specimens with positive ACE results for BVDV were immediately retested by performing immunohistochemistry (IHC). Follow-up testing, 3 to 4 weeks after initial positive ACE results, was done by use of a second IHC test and virus isolation on a subsequently submitted ear notch specimen from the same calves to identify those that were persistently infected (PI). RESULTS: 102 producers submitted ear notch specimens for BVDV screening. Initially, 24 of 12,030 calves had positive ACE results for BVDV. A second ear notch specimen was submitted for 20 of these 24 calves. Of 20 retested calves, 12 had positive ICH results for BVDV, confirming PI status. The 12 PI calves came from 4 calf groups (3 singletons and 1 calf group with 9 PI calves). CONCLUSIONS AND CLINICAL RELEVANCE: Prevalence of BVDV in calf groups was low, and questions designed to identify high-risk biosecurity behaviors had little value in identifying calf groups with positive results for BVDV.     

Fetal protection following exposure to calves persistently infected with bovine viral diarrhea virus type 2 sixteen months after primary vaccination of the dams

This study demonstrated that the bovine viral diarrhea virus (BVDV; types 1 and 2) fractions of a multivalent vaccine protected pregnant heifers and their fetuses at 149 to 217 days of gestation against exposure to calves persistently infected with BVDV type 2a. Eighty percent (eight of 10) of the control heifers were viremic at least 1 day following challenge, whereas all (20 of 20) BVDV-vaccinated heifers were virus isolation-negative on all postchallenge assessment days. Ninety percent (nine of 10) of the calves born to control heifers but only 5% (one of 20) of calves born to BVDV-vaccinated heifers seroconverted to BVDV type 2 before ingesting colostrum. One calf born to a control heifer was persistently infected. No calves from BVDV-vaccinated heifers were persistently infected.     

Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus

Eight healthy cattle that were persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) were inoculated with cell culture fluids that contained noncytopathic or cytopathic BVDV. A severe disease occurred after inoculation with cytopathic BVDV. The clinical signs, lesions, and immune response were consistent with those of clinical BVDV infections.     

Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus

Nine steers persistently infected with noncytopathic bovine viral diarrhea (BVD) virus were allotted into 3 groups (3 cattle/group). Cattle in group A were vaccinated with a modified-live BVD virus vaccine of porcine cell origin, cattle in group B with a modified-live BVD virus vaccine of bovine cell origin, and cattle in group C with a killed BVD virus vaccine of bovine cell origin. Detrimental effects due to vaccination were not seen. Six weeks after vaccination, the steers were challenge exposed with a cytopathic BVD virus. All steers developed mucosal disease after challenge exposure, produced antibodies that neutralized various isolates of BVD virus, and remained persistently infected until death. Steers given killed virus vaccine had a minimal neutralizing-antibody response and developed mucosal disease as quickly as reported for challenge-exposed, nonvaccinated, persistently infected cattle. Steers given modified-live virus vaccines had higher neutralizing-antibody response and longer intervals from challenge exposure to development of mucosal disease. The specificity of the neutralizing-antibody response differed between groups of vaccinated cattle.     

Effects of incubation temperatures and bovine viral diarrhea virus immune serum on bovine turbinate cells persistently infected with a noncytopathogenic isolate of bovine viral diarrhea virus

Noncytopathogenic bovine viral diarrhea virus (BVDV) on initial inoculation in receptive cells induced a persistence in vitro at incubation temperatures of 33, 37, and 39 C. The mechanisms of this persistence were not the result of the induction of defective interfering particles or the selection of temperature-sensitive mutants, but were the result of a naturally occurring noncytopathogenic isolate of BVDV. Persistently infected cells were not freed of the infection by continuous passage in media containing homologous antibody. Persistently infected cells appeared to undergo an earlier senescence than did noninoculated cells of the same passage level and age. This phenomenon was reversed by renewal of the culture media.     

Lymphocyte blastogenesis and neutrophil function in cattle persistently infected with bovine viral diarrhea virus

Neutrophil function and mononuclear cell proliferative responses to mitogens were determined in healthy cattle and in cattle persistently infected with bovine viral diarrhea (BVD) virus. Uptake of [3H]thymidine by resting and mitogen-stimulated peripheral blood mononuclear cells was significantly lower in cattle persistently infected with BVD virus than in healthy cattle. Neutrophils from cattle persistently infected with BVD virus had significantly impaired capability to ingest Staphylococcus aureus, but were normal in respect to random migration under agarose, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity. Impairment of neutrophil function in cattle persistently infected with BVD virus differs from impairment of neutrophil function reported in healthy cattle mounting an immune response to recent BVD virus infection.     

Viral and viral protein specificity of antibodies induced in cows persistently infected with noncytopathic bovine viral diarrhea virus after vaccination with cytopathic bovine viral diarrhea virus

Neutralizing and nonneutralizing antibodies to bovine viral diarrhea (BVD) virus were detected in 3 cows persistently infected with noncytopathic BVD virus after vaccination with modified-live cytopathic BVD virus. Neutralizing antibodies detected in serum samples from each persistently infected cow at 3 weeks after vaccination were highly specific for certain isolates of cytopathic BVD virus and reacted only with a viral protein with a molecular weight of 53,000. Neutralizing antibodies to 1 of 3 isolates of noncytopathic BVD virus were detected in a serum sample obtained at 12 weeks after vaccination from 1 of 3 persistently infected cows. Nonneutralizing antibodies were detected in all cows at 7 to 12 weeks after vaccination. The nonneutralizing antibodies were less specific for isolates of BVD virus and reacted with viral proteins with molecular weights of 115,000, 80,000, 53,000, and 47,000.     

Identification and eradication of bovine viral diarrhea virus in a persistently infected dairy herd.

A milking herd consisting of 55 Holstein cows had experienced abortions in several cows, as well as congenital malformations in 1 newborn calf. Bovine viral diarrhea virus was isolated from blood mononuclear cell samples obtained from several cattle, documenting 1 acute infection and 8 persistently infected carriers identified by clinical appearance and laboratory testing. Initial suspicion of persistently infected status in some, but not all animals, was facilitated by poor growth rates in some calves. Virus isolation was performed on transtracheal wash fluid obtained from acutely and persistently infected cattle with respiratory tract infection. We describe the measures taken to identify and characterize the infecting virus strain, and the series of actions taken to identify and eliminate persistently infected carriers in a herd experiencing several related problems that were shown to be caused by bovine viral diarrhea virus.     

Study of cattle persistently infected with bovine viral diarrhea virus that lack detectable virus in serum

OBJECTIVE: To determine whether cattle persistently infected with bovine viral diarrhea virus (BVDV) that lack virus detectable in serum by use of the immunoperoxidase microtiter assay (IPMA) can transmit the virus to susceptible herdmates and determine prevalence of these cattle. DESIGN: Clinical trial and serologic survey. SAMPLE POPULATION: 2 cattle and 1,952 blood samples. PROCEDURE: A persistently infected cow in which virus could not be detected in serum was housed with a BVDV-seronegative steer. Blood and nasal swab specimens were tested via virus isolation and serum virus neutralization. Parallel WBC preparations and sera from blood samples of 1,952 adult cows were screened for BVDV by use of IPMA. RESULTS: The steer seroconverted to BVDV within 4 weeks of contact with the cow. Virus was detected in sera and WBC of 5 adult cows that were verified as persistently infected by retest 3 weeks later. Cattle persistently infected with BVDV in which virus could not be detected in both serum and WBC by use of IPMA were not found. CONCLUSION AND CLINICAL RELEVANCE: Cattle persistently infected with BVDV in which virus cannot be detected in serum by use of IPMA may serve as virus reservoirs for infecting susceptible cattle. Persistent infection was detected at a prevalence of 0.26%. Screening adult cattle by use of IPMA on serum samples appears to be a reliable means of detecting persistent infection with BVDV. Prevalence of cattle persistently infected with BVDV that have negative results of IPMA on serum is extremely low.     

Transmission of Bovine viral diarrhea virus 1b to susceptible and vaccinated calves by exposure to persistently infected calves

Bovine viral diarrhea virus (BVDV) persistently infected (PI) calves represent significant sources of infection to susceptible cattle. The objectives of this study were to determine if PI calves transmitted infection to vaccinated and unvaccinated calves, to determine if BVDV vaccine strains could be differentiated from the PI field strains by subtyping molecular techniques, and if there were different rates of recovery from peripheral blood leukocytes (PBL) versus serums for acutely infected calves. Calves PI with BVDV1b were placed in pens with nonvaccinated and vaccinated calves for 35 d. Peripheral blood leukocytes, serums, and nasal swabs were collected for viral isolation and serology. In addition, transmission of Bovine herpes virus 1 (BHV-1), Parainfluenza-3 virus (PI-3V), and Bovine respiratory syncytial virus (BRSV) was monitored during the 35 d observation period. Bovine viral diarrhea virus subtype 1b was transmitted to both vaccinated and nonvaccinated calves, including BVDV1b seronegative and seropositive calves, after exposure to PI calves. There was evidence of transmission by viral isolation from PBL, nasal swabs, or both, and seroconversions to BVDV1b. For the unvaccinated calves, 83.2% seroconverted to BVDV1b. The high level of transmission by PI calves is illustrated by seroconversion rates of nonvaccinated calves in individual pens: 70% to 100% seroconversion to the BVDV1b. Bovine viral diarrhea virus was isolated from 45 out of 202 calves in this study. These included BVDV1b in ranch and order buyer (OB) calves, plus BVDV strains identified as vaccinal strains that were in modified live virus (MLV) vaccines given to half the OB calves 3 d prior to the study. The BVDV1b isolates in exposed calves were detected between collection days 7 and 21 after exposure to PI calves. Bovine viral diarrhea virus was recovered more frequently from PBL than serum in acutely infected calves. Bovine viral diarrhea virus was also isolated from the lungs of 2 of 7 calves that were dying with pulmonary lesions. Two of the calves dying with pneumonic lesions in the study had been BVDV1b viremic prior to death. Bovine viral diarrhea virus 1b was isolated from both calves that received the killed or MLV vaccines. There were cytopathic (CP) strains isolated from MLV vaccinated calves during the same time frame as the BVDV1b isolations. These viruses were typed by polymerase chain reaction (PCR) and genetic sequencing, and most CP were confirmed as vaccinal origin. A BVDV2 NCP strain was found in only 1 OB calf, on multiple collections, and the calf seroconverted to BVDV2. This virus was not identical to the BVDV2 CP 296 vaccine strain. The use of subtyping is required to differentiate vaccinal strains from the field strains. This study detected 2 different vaccine strains, the BVDV1b in PI calves and infected contact calves, and a heterologous BVDV2 subtype brought in as an acutely infected calf. The MLV vaccination, with BVDV1a and BVDV2 components, administered 3 d prior to exposure to PI calves did not protect 100% against BVDV1b viremias or nasal shedding. There were other agents associated with the bovine respiratory disease signs and lesions in this study including Mannheimia haemolytica, Mycoplasma spp., PI-3V, BRSV, and BHV-1.     

Reproductive performance of apparently healthy cattle persistently infected with bovine viral diarrhea virus.

A Holstein-Friesian bull and three Holstein-Friesian cows were seronegative for bovine viral diarrhea (BVD) virus but were persistently infected with the virus. Virus was isolated from buffy coat cells and nasal and lacrimal secretions during their lifetime, and they remained free of clinical signs of BVD. The three cows were pregnant when purchased, and they gave birth to full-term calves. One calf lived only a few hours, one calf became ill and died within a few days, and one calf became ill and was euthanatized within a few weeks. One cow was then bred and became pregnant but aborted a 7-month fetus. A second cow was bred approximately 5 months after parturition but did not conceive. The third cow was necropsied 6 weeks after calving, because of loss of weight. Although the bull's semen contained BVD virus when seropositive cows were bred, normal calves were born. When seronegative heifers were bred, they became seropositive to BVD virus within two weeks, with higher titers in six weeks. On heifer conceived after one service but aborted a 6-month fetus. Three others continued to have estrous cycles until their titers rose to 1:128, then they conceived and gave birth to normal calves. Another heifer conceived on the first service, had a titer of 1:128 two weeks after breeding, and gave birth to a normal calf.     

Association between the existence of calves persistently infected with bovine viral diarrhea virus and commingling on pen morbidity in feedlot cattle

OBJECTIVE: To determine the association between the existence of a calf persistently infected (PI) with bovine viral diarrhea virus (BVDV) and pen morbidity. ANIMALS: 5,041 calves in 50 pens at a feedlot in Iowa. PROCEDURE: In a longitudinal study, ear notches were collected from cattle and tested for BVDV antigen. Characteristics of each pen (owner, sex, disease rate, number of groups, and source) were recorded. The association between the existence of a BVDV-PI calf and morbidity in each pen was examined. RESULTS: Commingling was associated with an increase in respiratory tract disease (odds ratio [OR], 3; 95% confidence interval [CI], 2.5 to 3.6). Ten BVDV-PI calves (10/5,041 [0.2%]) were identified in 8 of 50 pens. A BVDV-PI calf was associated with reduced pen-level respiratory tract disease (OR, 0.7; 95% CI, 0.5 to 0.9). Disease prevalence (mean +/- SD morbidity, 7.9 +/- 3.1%) was lowest in pens containing single-source cattle and a BVDV-PI calf (4 pens containing 302 cattle), compared with single-source cattle with no BVDV-PI calf (mean morbidity, 11.89 +/- 9.7%; 31 pens containing 3,093 cattle), commingled cattle with no BVDV-PI calf (mean morbidity, 29.3 +/- 16.22%; 11 pens containing 1,127 cattle), and commingled cattle with a BVDV-PI calf (mean morbidity, 28.6 +/- 10.1%; 4 pens containing 519 cattle). CONCLUSIONS AND CLINICAL RELEVANCE: Commingling was the greatest risk factor associated with morbidity in each pen. A BVDV-PI calf in a pen was not associated with increased disease prevalence in commingled groups.     

Fatal trichuris spp. infection in a Holstein heifer persistently infected with bovine viral diarrhea virus

Whipworms (Trichuris spp.) were identified in the colon of a recently purchased, 10-month-old dairy heifer that died suddenly. A skin test was positive for bovine viral diarrhea virus (BVDV). Signs of BVDV occurred in other heifers in the group, but fecal flotations were negative for whipworm eggs.