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Canadian Journal of Veterinary Research logoLink to Canadian Journal of Veterinary Research
. 2020 Jul;84(3):163–171.

Infection of calves with in-vivo passaged bovine parainfluenza-3 virus, alone or in combination with bovine respiratory syncytial virus and bovine coronavirus

John Ellis 1,✉, Nathan Erickson 1, Sheryl Gow 1, Keith West 1, Stacey Lacoste 1, Dale Godson 1
PMCID: PMC7301673  PMID: 32801450

Abstract

Bovine respiratory disease complex is etiologically complex and usually involves co-infection by several agents, including bovine parainfluenza virus-3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine coronavirus (BCoV). Traditionally, vaccines have been tested in seronegative calves infected with a single in vitro-passaged agent, often with little disease, resulting in unvaccinated subjects. To overcome the potential problem of attenuation coincident with in vitro culture of the viruses, cocktails of field isolates of BPIV-3s and BCoVs were passaged in the lungs of neonatal colostrum-deprived calves. Lung lavage fluids were used as inocula, alone and in combination with in-vivo passaged BRSV, and aerosolized into a trailer containing conventionally reared 9-week-old weaned Holstein calves with decayed, but still measurable, maternal antibodies. Calves developed acute respiratory disease of variable severity. Upon necropsy, there were characteristic gross and histologic lesions in the respiratory tract, associated immunohistochemically with BPIV-3, BRSV, and BCoV. In-vivo passage of viruses is an alternative to in vitro culture to produce inocula to better study the pathogenesis of infection and more rigorously and relevantly assess vaccine efficacy.

Introduction

It is long-established that respiratory disease in cattle is multifactorial, etiologically complicated, and a result of mixed infections; hence the term, bovine respiratory disease complex (BRDC) (1). From a virological perspective, the common occurrence of co-infections was historically documented with culture (2), whereas now, it is done with PCR and metagenomic analyses (3,4). Nevertheless, the experimental approach to BRDC has largely been reductionist, usually employing single agent challenge models. Furthermore, high doses of cultured pathogens are often given by unnatural routes, such as trans-tracheal injection of seronegative calves, which may be irrelevant in the real world. Arguably, this has been driven by regulatory agencies, which require experimental conditions free of confounders for vaccine licensure; the result is a wedding to a literal reading of Koch’s Postulates: one agent, one disease.

Bovine parainfluenza-3 virus (BPIV-3) is one of the “original” pathogens in the BRDC, having been first implicated etiologically in the syndrome in 2 simultaneous reports in 1959 (5,6). Since it was considered a primary pathogen in cattle, vaccine development commenced in the 1960s, shortly after its isolation from affected cattle; BPIV-3 has been a component in most combination bovine parenteral and intranasal vaccines since that time (7). In the interim, between its indictment in BRDC and the present, many veterinarians have come to view BPIV-3 as a relatively innocuous agent and some have questioned the necessity of including it in vaccines (7). There are at least 3 explanations that would appear to substantiate that impression. First, because of BPIV-3 endemicity and routine vaccination of cattle populations, there is good disease-limiting herd immunity to the virus (7). Second, acutely infecting respiratory paramyxoviruses have a relatively short life cycle in the respiratory tract (< 10 d), based primarily on experimental infections (7,8) (John Ellis, University of Saskatchewan, unpublished data 1997–2018). Reflecting this brevity, in the field, if etiologic diagnoses using immunohistochemistry are attempted on lungs of cattle that die of BRDC of more than 7 days duration, usually only bacteria, and rarely acutely infecting viruses such as BPIV-3, are found in either acute or chronic lesions (9). This can potentially result in false negative results, unless acute and convalescent sera are also examined. Finally, and most relevant to this study, it is empirically recognized, though poorly formally documented, that the respiratory paramyxoviruses BPIV-3 and bovine respiratory syncytial virus (BRSV), attenuate rapidly upon culture in vitro (7,8). This makes the development of vaccines relatively facile but complicates experimental testing efforts. In effect, virtually all commercially available BPIV-3 vaccines have been licensed based on challenge studies with repeatedly passaged, highly attenuated, and relatively avirulent cultured BPIV-3 viruses. These are essentially vaccine viruses, that because of practical and logistical concerns, regulatory agencies provide or pharmaceutical companies maintain in-house. A review of the literature on BPIV-3 vaccines (7) will validate the latter provocation; little if any clinical disease or respiratory pathology is usually reported in controls, with nasal shed of virus often being the primary (or only) outcome variable. Therefore, a review of the literature could suggest that BPIV-3 is a virus in want of disease, a component of most bovine combination vaccines with little utility.

Bovine respiratory syncytial virus was first serologically associated with respiratory disease in cattle in 1968 and first isolated from clinical cases in 1970 (8). Its endemicity in cattle populations and its importance as a respiratory pathogen, especially in calves, became well-established shortly thereafter, prompting vaccine development. By the early 1980s, both modified-live and inactivated parenteral vaccines were commercially available; intranasal vaccines were developed in the mid-2000s (10). Until the late 1990s, BRSV vaccines were evaluated and licensed using cultured BRSV in challenge models that produced little more than mild pyrexia and nasal shed of the virus. Since then, commonly used BRSV vaccines, at least in North America, have been more stringently tested in a disease-producing challenge model using low-dose aerosolized in-vivo passaged BRSV (10).

Bovine coronavirus (BCoV) was first implicated as a bovine pathogen in 1972 for its role in calfhood diarrhea (11). Ten years later, it was implicated as a respiratory pathogen in a study of agents in clinical samples from cases of calfhood pneumonia (2). Interestingly, in the latter seminal study, the difficulty in reproducing respiratory disease with BCoV or BRSV, or BPIV3 alone was noted, alluding to the complexity of BRDC (2). Since that time, the importance of BCoV as a respiratory pathogen has been somewhat controversial (12), at least in part because of the continuing difficulty in experimentally reproducing respiratory disease with cultured BCoVs; as early studies with the virus demonstrated that adaptation to growth in cell lines is attendant with attenuation. There have been several published attempts to reproduce respiratory disease with BCoV, with mixed results at best; however, none have used aerosolized in-vivo passaged virus (12).

Based on experience with BRSV (13), we hypothesized that in-vivo passaged BPIV-3 and BCoV would maintain the virulence of field isolates of the viruses and provide inocula that would more likely produce disease than in vitro-passaged viruses. Moreover, combinations of these 3 viral pathogens recognized in calfhood in a challenge model would better mimic the reality of mixed infections in calfrearing operations and provide a means to better evaluate the true efficacy (i.e., disease sparing) of vaccines containing these viruses.

Materials and methods

Calves

For the preparation of in-vivo passaged inocula, Holstein calves were removed from the damns at birth and deprived of colostrum (CD calves). They were given 1 mL of 2× enrofloxacin (Baytril; Bayer, Mississauga, Ontario) daily for 5 to 6 d and maintained on milk replacer (3 L twice daily) in individual biosecurity level 2 containment rooms.

For the 2 challenge experiments, Holstein calves were removed from their dams at birth and fed 2.1 L of a (reconstituted) commercial colostrum replacement product (Calf’s Choice Total; The Saskatoon Colostrum Company, Saskatoon, Saskatchewan) containing a total of 150 g of IgG that is high in BPIV-3, BRSV, and BCoV antibodies (14,15). Calves were given 1.5 mL of tulathromycin (Draxxin; Zoetis, Whitby, Ontario) subcutaneously at 2 to 3 d of age, and in the case of the BPIV3 challenge, 3 mL of a modified-live combination bovine coronavirus and bovine rotavirus vaccine (Calf-Guard; Pfizer Animal Health, Whitby, Ontario) intranasally. Calves were tested for bovine viral diarrhea virus (BVDV) (7) and reared as previously described in individual pens (15) until weaning at approximately 9 wk of age, when they were group housed after challenge.

Viral challenge inocula

BPIV-3

The BPIV-3 inoculum was an admixture of 6 low passage (1 to 3) archival isolates from field cases of respiratory disease in the late 1980s and early 1990s (Figure 1). At the time of the original isolation, all isolates tested positive for BPIV-3 by immunofluorescence-staining of cytospin preparations and negative for bovine herpesvirus-1 (BHV-1), BRSV, and BVDV. Aliquots of bovine embryonic cells (passage 6; 4 × 106 in 4.6 mL) were inoculated with 0.75 mL of one of the 6 isolates in 75 cm2 flasks. On day 5 post-inoculation, when approximately 100% of the cytopathic effect (including formation of syncytia and cell death) was observed, cultures were frozen at −80°C. Following rapid thawing, 4 mL of each expanded isolate was combined in a 50 mL centrifuge tube, which was swirled. The mixture was nebulized (Ultra-Neb 99; DeVilbiss, Somerset, Pennsylvania, USA) into the nostrils of a neonatal calf using a homemade mask that completely enveloped both nostrils. Five days later, the calf had developed signs of mild respiratory disease and was euthanized by barbiturate overdose. A whole lung lavage was performed in situ by inserting a canula mid-cervically into the exposed trachea, which resulted in an inoculum with a titer of 3 × 106 tissue culture infective dose50 (TCID50) units of BPIV-3/mL, which was stored at −80°C. Five years later, for a second in-vivo passage, 25 mL of the cryopreserved lung lavage was nebulized into another neonatal CD calf. Five days later, the calf developed signs of mild respiratory disease and was euthanized by barbiturate overdose. Another whole lung lavage was performed, resulting in an inoculum with a titer of 3 × 107.3 TCID50 BPIV-3/mL. This lavage fluid was confirmed negative for bacteria and Mycoplasma spp. by culture, and negative for BHV-1, BRSV, and BVDV by PCR. A low concentration of BCoV (estimated < 103 TCID50/mL; 31.59 Ct value) was detected by qPCR.

Figure 1.

Figure 1

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Schematic of in-vivo passage and challenge approach for BCoV, BPIV-3, and BRSV individually and combined.

BRSV and BPIV-3 combination

For preparation of an inoculum containing both BRSV and BPIV3, the latter 2nd passage BPIV3 inoculum (above, cryopreserved for 14 y) was diluted 1000-fold into 25 mL modified Eagles medium (MEM) and mixed with 25 mL of a 14th in-vivo passage of BRSV (Asquith strain; lot 04-17) to equilibrate the titers of each of the paramyxoviruses to approximately 104 TCID50/mL (Figure 1). The BRSV was previously confirmed negative for bacteria and Mycoplasma spp. by culture and negative for BHV-1, BVDV, and BCoV by PCR and immunohistochemistry of infected lung in the case of questionable suspect PCR results. Various in-vivo passages of this BRSV inoculum have produced consistent severe respiratory disease in naïve cattle (10). Forty mL of the combined BRSV/BPIV-3 mixture was nebulized into the nostrils of a neonatal CD calf using a mask (as described). On day 6 after challenge, the calf was euthanized by barbiturate overdose and a whole lung lavage was performed as described. The resulting inoculum contained approximately 103.5 TCID50/mL BRSV (Ct value = 22.06) and 102.1 TCID50/mL (Ct value = 21.32) BPIV-3, as estimated by comparison with standard curves of qPCR Ct values generated by diluting and testing known quantities of the respective viruses. No BCoV was detected by qPCR.

BCoV

The source of BCoV was from an outbreak of acute respiratory disease in approximately 6-month-old Angus-Simmental cross beef calves in the fall of 2003, the notable history being a consistent seroconversion to BCoV and lack of antibody response to BRSV or BHV-1. Nasal swabs were collected from affected calves and a cytopathic virus was isolated in human rectal tumor (HRT) cells; immunofluorescence staining revealed affected cells were positive for BCoV antigens. One mL of first passage and 4.5 mL of third passage isolates (each approximately 106 TCID50/mL) were added to 25 mL of MEM for the initial BCoV inoculum (Figure 1). This mixture was nebulized into the nostrils of a neonatal CD calf using a mask. On day 6 after challenge, a whole lung lavage was performed resulting in an inoculum containing approximately 104 TCID50/mL BCoV (Ct value = 22.06), which was estimated by comparing with a standard curve of qPCR Ct values generated by diluting a known amount of BCoV.

Challenges and clinical assessments

BPIV-3 challenge

On the day of weaning (approximately 9 wk of age), 14 calves were challenged as previously described for BRSV (15), but with minor modifications. They were challenged briefly by aerosol delivery of lung lavage fluid into an enclosed, approximately 5 × 2.5 × 2.5 m, transport (stock) trailer. For aerosol delivery, 40 mL of in-vivo passaged inoculum was placed in each of 3 ultrasonic nebulizers that were placed equidistant approximately 2 m off the floor of the trailer. After approximately 60 min in the sealed trailer, calves were removed from the trailer and maintained as a single group in 1 large covered pen (15).

BPIV-3-BRSV-BCoV combination challenge

On the day of weaning (approximately 9 wk of age), when calves had residual maternal antibodies, 3 were challenged as previously described for BRSV (15), but with minor modifications. They were briefly challenged by aerosol delivery of lung lavage fluid mixture into an enclosed, approximately 2.5 × 2.5 × 2.5 m, transport (horse) trailer. For aerosol delivery, 40 mL of in-vivo-passaged BRSV/BPIV-3 inoculum was mixed with 40 mL of in-vivo passaged BCoV inoculum and 40 mL of this mixture was placed in each of 2 ultrasonic nebulizers that were placed equidistant approximately 2 m off the floor of the trailer. After approximately 30 min in the sealed trailer, calves were removed from the trailer and maintained as a single group in 1 large covered pen (15).

In both above challenge experiments, calves were observed for clinical signs as previously described (Appendix) (15) on days 1 and 0 prior to challenge and on days 1 through 6 after challenge, with the respective inocula. Calves were euthanized on day 6 after challenge or earlier on the basis of predetermined criteria (15); 2 clinical signs indicative of substantial respiratory tract disease had to be observed, including moderate signs of depression, dull eyes, droopy ears, rough coat, gauntness, and moderate respiratory distress or dyspnea (> 100 breaths/min), for 2 consecutive days. Calves were euthanized immediately if they showed signs of severe respiratory distress (e.g., pronounced open-mouthed labored breathing, as evidenced by an expiratory grunt), if they were severely depressed and recumbent with total reluctance to rise, or if they had a PaO2 < 50 mmHg. These criteria were consistent with the Canadian Council of Animal Care guidelines that were approved by the Committee on Animal Care and Supply at the University of Saskatchewan and have been used routinely in efficacy studies of BRSV vaccines.

Sample collection

Jugular venous blood was collected for serum on the day of challenge and the final day of the trial. Arterial blood samples were collected once on day 6 from the caudal thoracic aorta (16) and PaO2 (mmHg) measurements, corrected for rectal temperature, were performed as previously described (16). Deep nasal swab specimens were taken daily from both nares before challenge and after challenge for virus isolation (17).

Quantitative virus isolation

Quantitation of BPIV-3, BRSV, and BCoV was done using microisolation plaque assays with bovine embryonic lung fibroblasts (BPIV-3, BRSV), HRT cells (BCoV), and BRSV (17), and/or using qPCR for the respective viruses (18–20). Real-time qPCR was performed using previously described primers and probes for the BPIV-3 matrix gene (18), the BRSV fusion gene (19), and the BCoV transmembrane gene (20). Viral RNA was extracted using an RNeasy Plus Mini Kit (Qiagen, Toronto, Ontario). A 25-μL reaction mixture was used, containing 15 μL containing primers (10 μL) and probe (5 μL), 1 μL of RT-PCR enzyme mix, 4.5 μL of RNase, DNase free water, and 5 μL of template RNA. Thermocycle conditions were as follows: RT step at 48°C for 30 min, enzyme activation at 95°C for 10 min, followed by 40 amplification cycles of 95°C for 15 s and 60°C for 60 s. To estimate replicating virus in inocula and nasal swabs, qPCR CT values were compared with CT values on curves derived from qPCR testing of dilutions of known (tittered by microisolation) quantities of the respective viruses.

Postmortem analyses

For the BPIV-3 challenge, at necropsy, the respiratory tract of each calf was harvested and analyzed for percentage of pneumonic tissue essentially as previously described (13), except that tracings were made from printed color images. For the BPIV, BRSV, and BCoV combination challenge, the percentage of pneumonic lung was visually assessed and quantitated using a weighted scoring rubric (21). Selected sections of trachea and lung were immunohistochemically stained for BPIV-3, BRSV, and/or BCoV antigens using relevant antibodies as previously described (22).

Antibody assays

The BPIV-3 neutralization tests (VNT) and BPIV-3-and BCoV specific IgG ELISAs for serum were performed and analyzed as previously described for BRSV, using BPIV-3 or BCoV and BPIV-3 antigens and relevant control sera (23).

Results

BPIV3 infection alone challenge

Calves developed one or more signs of respiratory disease, including pyrexia, tachypnea, dyspnea, and spontaneous coughing of variable severity, after aerosol challenge with BPIV-3; the prevalence and severity of which are indicated in Figures 2 to 5. Nasal discharge and depression were observed in only a few calves on a few days after infection and were not considered prominent clinical features of infection (data not shown).

Figure 2.

Figure 2

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Group mean rectal temperatures in 9-week-old weanling Holstein calves on days after infection with BPIV-3.

Figure 5.

Figure 5

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Spontaneous coughing in 9-week-old weanling Holstein calves on days after infection with BPIV-3.

None of the calves had detectable BPIV-3 on nasal swabs on the day of weaning/challenge (< 10 TCID50/mL). Beginning on day 1 after infection, BPIV-3 was detectable in nasal secretions; 8 of 14 calves had ≥ 2 × 102 TCID50/mL. All 14 calves shed (> 20 PFU/mL) on days 2 to 6 after infection. In most wells of the microisolation plates, there was diffuse staining indicative of a high amount of virus > 104 TCID50/mL with the number of plaques too numerous to count.

Thirteen of the 14 infected calves had pO2 concentrations below the normal range for cattle [standard deviation (SD) ±16; range: 80 to 100 mmHg] on day 6 after challenge. The mean pO2 was 64.3 mmHg (SD ±11.1; range: 45 to 93.7 mmHg). The lungs of all the calves had lesions of variable severity that consisted of sunken plum-colored areas of atelectasis that were most prominent in the anterior ventral regions of the lung, which is characteristic of acute BPIV-3 infection (Figure 6). The mean percentage of the pulmonary lesional area was 23.5% (SD ±7.9; range: 10.0% to 32.5%). Histologically, BPIV-3 was demonstrated by immunohistochemistry in a standard section distal to the hilar region of the left middle lobe in characteristic lesions of bronchointerstitial pneumonia, including necrotizing bronchiolitis and alveolitis, with intracytoplasmic inclusion bodies and an accompanying mixed inflammatory cell infiltrate, in the lungs of all 14 calves. Viral antigen was present in airway epithelial cells, pulmonary parenchyma (type I epithelium), and cells with the morphology of pulmonary alveolar macrophages. Viral antigen was also visible in frequent syncytia and in common variably sized intracytoplasmic inclusion bodies (Figures 7, 8). Bovine coronaviral antigens were only found in a few scattered mucosal columnar epithelial cells in tracheas, but not in pulmonary airways or parenchyma.

Figure 6.

Figure 6

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Lungs from a 9-week-old weanling Holstein calf euthanized on day 6 after infection with BPIV-3. Note the sunken plum-colored areas of atelectasis.

Figure 7.

Figure 7

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Low power (4×) micrograph of a section of an atelectatic focus from a lung from a 9-week-old weanling Holstein calf euthanized on day 6 after infection with BPIV-3. Note the brown precipitate indicative of BPIV-3 antigens in epithelial cells in large and small airways and pulmonary parenchyma. Immunohistochemical staining for BPIV-3.

Figure 8.

Figure 8

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High power (40×) micrograph of a section of an atelectatic focus from a lung from a 9-week-old weanling Holstein calf euthanized on day 6 after infection with BPIV-3. Note the brown precipitate indicative of BPIV-3 antigens in epithelial cells, including syncytial cells, in the terminal airway and pulmonary parenchyma. Note the common intracytoplasmic inclusion bodies. Immunohistochemical staining for BPIV-3.

All the calves had moderate to high concentrations of serum (maternal) antibodies (MatAb) to BPIV-3 after feeding of the colostrum replacement product (data not shown). By 9 wk of age, BPIV-3-specific MatAb had decayed [geometric mean titer (GMT) = 1/60; range = 1:18 to 1:162] but were still considered moderate concentrations. There was no detectable (anamnestic) antibody response subsequent to the BPIV-3 challenge, indicating that the calves had not been primed by natural exposure prior to challenge (data not shown).

BPIV3/BRSV co-infection in-vivo passage/ challenge

The CD calf simultaneously infected with equivalent amounts of BRSV and BPIV-3 developed mild respiratory disease on days 5 to 6 after challenge, including mild tachypnea and an increased rectal temperature of 39.5°C on day 6. The lungs failed to collapse completely upon opening the thoracic cavity. There were multifocal, disseminated (to all lobes), often jagged linear plum-colored foci (interpreted as atelectasis). Immunohistochemical staining of the trachea revealed numerous BRSV and/or BPIV-3-positive epithelial cells. In serial sections of a typical atelectatic focus, copious BRSV and BPIV-3 antigens were visible, both in epithelial cells lining airways and in the pulmonary parenchyma, including syncytial cells and intracytoplasmic inclusion bodies. It appeared that the same cells often contained both BRSV and BPIV-3 antigens (Figures 9, 10). No coronaviral antigens were detected in immunohistochemically stained sections of lung or trachea.

Figure 9.

Figure 9

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Medium power (10×) micrograph of a section of an atelectatic focus from a lung from a 6-day-old colostrum-deprived Holstein calf euthanized on day 6 after infection with BPIV-3 and BRSV. Note the brown precipitate indicative of BPIV-3 antigens in epithelial cells in large and small airways and pulmonary parenchyma. Immunohistochemical staining for BPIV-3.

Figure 10.

Figure 10

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Medium power (10×) micrograph of a serial section (from Figure 9) of an atelectatic focus from lung from a 6-day-old colostrum-deprived Holstein calf euthanized on day 6 after infection with BPIV-3 and BRSV. Note the brown precipitate indicative of BRSV antigens in epithelial cells in large and small airways and pulmonary parenchyma. Immunohistochemical staining for BRSV.

BCoV infection alone in-vivo passage/challenge

The CD calf infected with BCoV had voluminous mucoid and occasionally slightly blood-tinged feces on days 2 to 5 after infection. Aside from a moderate amount of crusted nasal discharge on day 5, there were no overt signs of respiratory disease and the calf was otherwise clinically normal with a good appetite. The trachea contained scant mucopurulent exudate, but there were no gross pulmonary lesions. Coronaviral antigens were observed in columnar epithelial cells in proximal, middle, and distal (from the larynx) sections of trachea (Figure 11). Coronaviral antigens were not observed in immunohistochemically stained sections of lung, ileum, or spiral colon.

Figure 11.

Figure 11

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Medium power (20×) micrograph of a mid-section of trachea from a 6-day-old colostrum-deprived Holstein calf euthanized on day 6 after infection with BCoV. Note the brown precipitate indicative of BCoV antigens in columnar epithelial cells and mild mixed inflammatory cell infiltrate in subjacent lamina propria. Immunohistochemical staining for BCoV.

BPIV3/BRSV/BCoV co-infection challenge

Three colostrum-fed calves that were challenged on the day of weaning (approximately 9 wk of age) by low dose aerosolization of a mixture of BPIV-3, BRSV, and BCoV developed clinical respiratory disease characterized by pyrexia (≥ 39.5°C), increased respiratory rates, mild to moderate dyspnea, and occasional coughing. Aortic oxygen concentrations of 61.3, 75.8, and 79.2 mmHg were just below or well below normal values (normal range: 80 to 100 mmHg) On day 6 after challenge, all calves had low to moderate amounts of all 3 viruses (approximately 2 to 3 log10 TCID50/mL) on nasal swabs, as determined by qPCR. Multiple plum-colored sunken irregular areas of atelectasis were present in lungs, with an overall percentage of lung lesional area in the 3 calves of 15.6%, 9.5%, and 23.3%, respective to the pO2 concentrations.

Discussion

To our knowledge, this is the first report on the use of in-vivo passaged BPIV-3 to reproduce the typical clinical disease and characteristic lesions that were first described in the initial reports of BPIV-3-associated respiratory disease in cattle (5,6) and later, in naturally occurring outbreaks (24,25). As in previous descriptions, interstitial emphysema, often with the formation of bullae in the parenchyma and sub-pleurally, was not a pathological finding in these calves, despite the presence of extensive bronchointerstitial pneumonia with widespread replication of BPIV-3 in airway and parenchymal epithelium; a clear difference with the typical lesions reported in naturally acquired and experimentally induced cases of BRSV infection (8,13). Relatedly, the pronounced expiratory dyspnea characteristic of BRSV infection (8,13,15) was mostly absent in these BPIV-3 infected calves. The basis for these apparent differences in pathogenesis is unresolved but may have to do with a more pronounced effect of BRSV on airway hyper-responsiveness (26).

Shortly after its isolation in the late 1950s, there were several attempts to reproduce typical clinical disease and lesions originally described in field cases of BPIV-3 infection (7). All of these used cultured virus in small numbers of colostrum-deprived calves and only the 2 (27,28) that used multiple and invasive (transtracheal) routes of administration resulted in significant clinical disease and pulmonary lesions. In the late 1970s, there was a report documenting production of clinical disease and significant lesions in colostrum-deprived calves that were inoculated intranasally with 10 mL of high titer (107) BPIV-3 (29); however, virtually all published assessments of vaccine efficacy reported minimal (if any) clinical disease and no examination/ documentation of pulmonary pathology in unvaccinated controls (7). In fact, assumedly due to the difficulty in consistently reproducing disease, seroconversion and reduced nasal shed are the only codified criteria for vaccine licensure (30), but arguably, represent a low bar for clinical protection. In contrast, the use of in-vivo passaged virulent virus provides a challenge method, low dose group aerosolization, that more closely mimics natural transmission that would occur in housing of calves or during transport, and therefore, is more relevant to assessing vaccine efficacy.

Given the low dose (< 3 logs10) of BCoV “contaminating” the BPIV-3 inoculum, which contained > 4 logs10 more virus/mL, it is unlikely that BCoV contributed significantly biologically to the disease observed in the BPIV-3 infection; BCoV was absent from the lungs and only found rarely in the tracheas in immunohistochemically stained sections. It is most likely, although not confirmed during the original isolation of the BPIV-3, that this BCoV derived from one or more of the BPIV-3 field isolates, which would be consistent with historic and current evidence of common viral co-infections in naturally-occurring BRDC (2,3). Historically, BCoV has been relatively difficult to isolate from field cases unless HRT cells are used (12). The use of embryonic lung cells, likely primarily fibroblasts, probably selected against isolation of BCoV. This would explain a resultant low concentration of BCoV in the BPIV-3 inoculum. It is also possible that there were other uncultured agents in the in-vivo passaged inocula that were not specifically identified in this study. Recently, metagenomics analyses have identified multiple viruses in bovine respiratory samples that have not been traditionally associated with BRD (4,31). The role of many of these agents in BRD is uncertain and their indictment will require prospective controlled studies, like the ones that have recently been conducted with influenza D (32). Regardless, the presence of copious amounts of BPIV-3 and BRSV in affected target cells in lesions and the disease-sparing from vaccine use (7,10), are evidence of the causal role of at least these agents in the disease observed.

In the case of BPIV-3/BRSV co-infection, it was perhaps not surprising that, given the overall similarities in the biology of these 2 respiratory paramyxoviruses (7,8), the same population of cells were infected by both viruses, including apparent common co- infection of the same cell by both viruses. Interestingly, although virtually the same titer (approximately 4 log10/mL) of each virus was used to infect the CD calf, the concentration of BPIV-3 in the resultant lung wash was approximately 10-fold less than that of BRSV (based on estimation by qPCR). This implies a competitive advantage in replication for BRSV versus BPIV-3, at least in the context of co-infection, which may be related to a real difference in comparative pathogenicity in vivo; lower viral load, less disease. To our knowledge, concurrent infection with BRSV and BPIV-3 has not been previously examined experimentally in vivo. Again, a basis for the apparent differences in pathogenesis of this aspect of the infections (i.e., relative replication) is unresolved.

Members of the Mononegavirales and Nidovirales orders of viruses exist as quasispecies, comprising viruses with different biological properties, including virulence (33). Currently, there are 3 recognized genotypes of BPIV-3, but little is known beyond that regarding any phenotypic differences (34). Genotypic and phenotypic analyses related to differences in virulence among BCoVs are also limited (12). Therefore, in order to maximize the chances of identifying virulent populations of viruses while minimizing the use of animals, we took an empirical approach and used cocktails of BPIV-3s and BCoVs that were isolated from field cases of respiratory disease. We allowed the infected calf to “select” which viruses in the quasispecies were more fit to replicate in the respiratory tract (trachea and lung). Extrapolating from what is known about BRSV, we assumed that (rapidly) increased viral load would be associated with lesions and clinical disease (8).

The clinical disease and lesions observed in the calves that were infected with the combination of BPIV-3, BRSV, and BCoV were typical, but considered mild to moderate. These initial experiments erred on the side of not overwhelming the subjects with high doses of 3 viruses administered simultaneously. Nevertheless, given previous results with BRSV and the results of the BPIV-3 infection alone presented herein, which used a significantly higher dose (> 4 log10 higher) than in the combination challenge, it is almost certain that increasing the dose of the challenge viruses in a combined infection would increase the severity of disease and resultantly, increase the stringency of any assessments of vaccination.

In our other model of bovine paramyxoviral respiratory disease involving BRSV (13), which has been used extensively to test the efficacy of a variety of vaccines (10), calves are routinely fed BRSV-antibody low colostrum that is identified by screening samples from many individual cows. This practice avoids the usual significant negative health effects of total failure of passive transfer subsequent to colostrum deprivation, as well as provides colostral factors that contribute to the maturation of the neonatal immune system (35); in other words, provision of a more relevant experimental subject. Except for the calves used to produce inocula (neonatal CD calves), the studies herein were performed in calves that were fed a uniform source of colostrum that contained high concentrations of antibodies to BPIV-3, BRSV, and BCoV, in part, since it was not possible to identify BPIV-3 or BCoV antibody-low colostrum in the same large population of cows from which BRSV antibody-low colostrum was identified (John Ellis, University of Saskatchewan, unpublished data 1999–2004); in other words, calves that were more typical real-world candidates for vaccination. Moreover, this demonstrated that significant disease could be produced in approximately 2-month-old calves with decayed concentrations of MaAb using in-vivo passaged BPIV-3 or a combination of in-vivo passaged BPIV-3, BRSV, and BCoV, which is consistent with previous work using in-vivo passaged BRSV alone in normal colostrum fed calves with decayed MaAb (15). Arguably, these models employing in-vivo passaged still virulent viruses in combination in calves fed normal colostrum, are most relevant for vaccine efficacy testing, since most vaccines vaccinated calves in the field have similar concentrations of MaAb at the times of vaccination and challenge and most calves are exposed to multiple pathogens.

Figure 3.

Figure 3

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Respiratory rates (breaths/min) in 9-week-old weanling Holstein calves on days after infection with BPIV-3.

Figure 4.

Figure 4

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Dyspnea scores in 9-week-old weanling Holstein calves on days after infection with BPIV-3.

Acknowledgments

The authors acknowledge the expert technical support of Ms. Lori Hassard in the original isolation of some of the viruses used in this study. Supported by University of Saskatchewan discretionary funds viral immunology (JE). The first author (JE) has conducted and conducts vaccine efficacy studies for Pfizer Animal Health, Zoetis, Merial, Merck Animal Health, Fort Dodge Animal Health, Boehringer Ingelheim Vetmedica, Elanco.

Appendix.

Clinical scoring

Rectal temperatures

  • 0 = < 103°F (39.4°C)

  • 1 = ≥ 103°F (39.4°C)

Depression

  • 0 = normal

  • 1 = mild; moves slowly, head down

  • 2 = moderate; tends to lie down, staggers

  • 3 = severe; recumbent or stands with difficulty

Estimated respiratory rate in breaths/min (BPM)

  • 0 = ≤ 44

  • 1 = 45 to 64

  • 2 = 65 to 80

  • 3 = ≥ 81

Dyspnea

  • 0 = normal

  • 1 = mild; short and rapid

  • 2 = moderate; labored, abdominal

  • 3 = severe; very labored, grunting

Cough (cough scores were assigned to calves with spontaneous coughing during the clinical exam observation period; approximately 1 h per day)

  • 0 = < 3 episodes

  • 1 = 3+ episodes

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