It was done by Gram stained smear for detection of pus cells and microorganisms. Quantitative culture of NPA specimens of patients and control was done. Undiluted and tenfold serial dilutions were inoculated with a calibrated loop on chocolate agar, blood agar and MacConkey agar plates, after over night incubation in appropriate conditions, Quantitative NPA cultures were considered positive when the growth of 106 (cfu/ml) or more was observed.

Nasopharyngeal aspirates were collected from patients and control group according to Svensson et al.,. Sterile normal saline solution was instilled in one nostril while occluding the other nostril, using a sterile blunt-tipped disposable syringe. Then the patient was instructed to forcibly exhale through the lavaged side into a sterile specimen cup. The sequence was then repeated in the other side of the nose. NPA specimens were examined microbiologically immediately and part of the specimens was stored in aliquots at -70°C for PCR. Two ml blood samples from both patients and control were collected into vacutainer, centrifuged and serum was separated and stored at - 20°C for HBoV-IgM antibodies by ELISA.

All serum samples were tested for BHV-1 antibodies using a commercial BHV-1 gB ELISA test kit, HerdChek* (IDEXX, Switzerland) having 100% sensitivity and 99.8% specificity. Suspect antibody test results (samples with blocking % greater than or equal to 45% but less than 55%) were considered as positive in the data analysis.

The herd BVDV status was established by testing up to 10 serum samples from randomly selected animals, at ages from six months up to age at first calving, for BVDV antibodies as recommended by Houe et al.. This enabled detection of a minimum prevalence of 20-28% depending on herd size. The PrioCheck BVDV Ab test kit (Prionics AG, Switzerland) was used for antibody testing. The test has a relative sensitivity and specificity of approximately 98% and 99%, respectively, compared to a virus neutralization test.

The herd BRSV status was established by testing up to 20 (depending on herd size) randomly selected serum samples from heifers for BRSV antibodies to allow detection of at least a 15% prevalence of BRSV antibody carriers in the herd at a 95% confidence level assuming 94.6% sensitivity and 100% specificity of the test. For BRSV antibodies, the Svanovir ELISA test (Svanova Biotech AB, Sweden) was used.

Depending on herd size up to 25 heifers and 10 cows were tested for Mycoplasma bovis antibodies in each herd. This enabled to detect the prevalence of at least 15% among heifers and 27% among cows with 95% level of confidence. BIO K 260 ELISA test (Bio-X Diagnostics, Belgium) with sensitivity and specificity of 100% in 10% cut-off of optical density of the positive control was used to measure M. bovis antibodies.

Serum samples were analyzed by indirect enzyme-linked immunosorbent assays (ELISA) according to the manufacturer’s instructions. This ELISA was done using INgezim antibody test kits for BVDV, BRSV, and BHV-1 (Ingenasa, Madrid, Spain), and Monoscreen ELISA BPI3 (Bio-X Diagnostics, Rochefort, Belgium). The results were read in a microplate photometer, where the optical density (OD) was measured at 450 nm. The cutoff was calculated as A = OD (corrected negative control). Nevertheless, results obtained were expressed as positive and negative based on the manufacturer’s recommended cutoff value for each pathogen.

Prevalence was determined by dividing the number of positive animals between the total animal of the sampled population. The results obtained were analyzed by the Chi-square test (χ2) to determine the statistical association between the variables, and the odds ratio (OR) was calculated to determine the probability of risk of the analyzed factors. Calculations were made using the SPSS Statistics for Windows, (IBM, USA) version 21.0.

Bovine clinical samples used in this study were submitted to KSVDL for routine diagnostic testing. The samples were obtained from naturally infected animals in the field by licensed veterinarians as a part of normal veterinary care and diagnostic investigations.

Standard post-mortem examinations were performed on infected animals (IG and EG), including macro- and micro-pathological examinations of the respiratory tissue. Lung tissue was prepared and cultured for the presence of bacteria and sections were stained with haematoxylin-eosin for histological investigation.

Indirect ELISAs for the detection of serum antibodies specific to BRSV (SVANOVIR® BRSV-Ab), BPIV-3 (SVANOVIR® PIV3-Ab) and BCoV (SVANOVIR® BCV-Ab) (all from Svanova Biotech AB, Uppsala, Sweden) were performed according to the manufacturer’s instructions. Sera collected from all calves every second day from D-2 to D60 were analysed for BRSV antibodies. In addition, sera were analysed for detection of antibodies against BCoV and BPIV-3 at specific time points: For IG on D-2 and D26, for EG on D26, D47 and D60, and for SG on D27, D47 and D60.

Calves (n = 44) with or without BRD (increased respiratory rate and/or dyspnoea) were sampled for diagnostic purposes. Sampling of the calves was granted an exemption from requiring ethics approval by the institutional Animal Experiment Commission “Dier Experimenten Commissie (DEC) Lelystad (2013111.b)” because sampling was performed for diagnostic purposes. BAL samples were obtained as described. Approximately 35–75 ml BAL was obtained from each calf after instillation of 100 ml PBS with 10% Fetal Calf serum (FCS). Foam, large purulent exudates and blood clots were removed from the BALF samples under aseptic conditions. BALF (25 mL) was centrifuged (4600×g, 10 min, 4 °C). Sediment was resuspended in 0.5 mL Dulbecco’s minimal essential medium (DMEM) with 5% FCS, carefully added to 1 mL freeze medium (DMEM, 50% FCS and 20% DMSO) and frozen at −80 °C. The BALF supernatants were also stored at −80 °C.

For testing the influence of centrifugation of BALF samples (4600×g, 10 min, 4 °C) on the PCR results we tested three variants of BALF samples: without centrifugation, supernatant and pellet obtained after centrifugation (50 times concentrated). DNA was extracted from 200 μL aliquots of BALF samples. We used the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science), with the Total NA External_lysis” protocol (Version 2.11). With the MagNA Pure LC Total Nucleic Acid Isolation Kit) 32 samples can processed per run. In all runs a positive control (a mix of 1.4 × 106 cfu/mL M. bovis, 0.5 × 107 cfu/mL M dispar and 1.3 × 105 cfu/mL M. bovirhinis) and a negative water control (NTC) was included.

Clinical samples (nasal and pharyngeal swabs and lung tissue) from bovine respiratory disease submissions to KSVDL were screened by a BRDC PCR panel which detects BVDV, BHV-1, BRSV and bovine coronavirus (BCV). A total of 204 samples were screened. Samples were collected in years 2011–2014 from twelve states: Nebraska (n = 48), Kansas (n = 112), Colorado (n = 6), Missouri (n = 1), Mississippi (n = 7), Texas (n = 4), Oklahoma (n = 2), Idaho (n = 2), Montana (n = 6), Oregon (n = 4), Washington (n = 11) and Virginia (n = 1). Samples were also screened for influenza D virus (IDV) using quantitative real time reverse transcription PCR as previously described. A 5’-nuclease reverse transcription PCR assay was designed to detect bovine rhinitis viruses targeting the 3D polymerase gene: probe, 5’-FAM-CGG CAG TCC AGG TCC AGT GT-Iowa Black-3’; Forward: 5’-CTT TTC GGT GTG ATT GGC AG-3’; Reverse: 5’-GAA ATC TAT CAG GGC AGG TCT G-3’. Viral RNA was extracted using the MagMAX-96 viral RNA isolation kit (Life Technologies) according to the manufacturer’s instructions. Real time reverse transcription PCR was performed using Qiagen Quantitect RT-PCR with BRV primers and probe as follows: 50°C, 30 minutes; 95°C, 15 minutes; followed by 40 cycles of 94°C for 15 seconds and 60°C for 60 seconds. The PCR assay specificity was confirmed using bovine rhinitis virus positive samples as determined by metagenomic sequencing as well as with cultures of common BRDC pathogens BVDV, BHV-1, BRSV, BCV, Mannheimia haemolytica, Histophilus somni, Pasteurella multocida and Mycoplasma bovis.

The chi-square tests were employed to compare the BRSV, BoHV-1 and BVDV-1 seropositivity and also with “age group”. Fisher’s exact test was used to compare BRSV status according to the expected prevalence of 80% (≥80%, “high”; < 80%, “low”) and the other variables. Only the variables with p < 0,2 (two-tailed Fisher) were analyzed by a logistic regression model. The analyses were performed using the Epi Info™ program v. 7.0.

For determining the diagnostic specificity, BALF samples were analysed with the PCR/DGGE method by the Animal and Plant Health Agency (APHA, Mycoplasma Team, Addlestone Surrey, UK) as earlier described [18, 25]. To determine the analytical sensitivity of the PCR/DGGE analysis, four 10-fold serial dilutions of M. bovis (7 × 104 cfu/mL), M. dispar (16 × 104 cfu/mL), and M. bovirhinis (0.5 × 104 cfu/mL), were prepared in PBS. Samples were sent to the APHA and analysed using the PCR/DGGE method.

Renilla-GFP RSV (1*10∧5 plaque forming units (PFU)) was pre-incubated for 1 hour at 37°C with 40 µl RPMI (10% FCS and 1% pen/strep) in the presence or absence of antibodies and then cooled for 15 min on ice. Pre-cooled PMN (1.5*10∧5 cells) and 40 µl RSV-Ig mixture was added and allowed to bind for 15 min in 96-wells plates. Plates were washed and incubated for 15 min at 37°C in pre-warmed RPMI. After washing, cells were treated with trypsin for 10 min on ice to remove extracellular RSV. Cells were incubated twice (30 sec) with acid medium (RPMI pH 2.5, 0.1% FCS) to remove FcR bound RSV-Ig, with a 1 min centrifugation step (1500 rpm) in between and afterwards. Finally, cells were washed and fixed with 1% PFA in PBS and GFP expression was analysed by flow cytometry.

HEp2 cells (5*10∧4 cells) cultured in IMDM (10% FCS, 1% pen/strep) were seeded overnight in flat-bottom 96-wells plates. Renilla-GFP RSV (1*10∧5 PFU) was pre-incubated for 1 hour at 37°C with 100 µl medium (1% FCS) in the presence or absence of antibodies and then added to the cells. Cells were trypsinised after 18–24 hour incubation and GFP expression was analysed by flow cytometry.

The value of diagnostic tests in calf pneumonia is somewhat limited due to the multifactorial nature of the disease and the uncertainty if the pathogens recovered from samples are causative to the disease. Most outbreaks can be successfully managed using the principles for treatment described above, and diagnostic tests cannot replace the examination of management practices and facility design in cases of recurrent outbreaks.

If animals are selected for ante-mortem sampling they should be in the early stages of the pneumonic process, before treatment, and should show typical signs of the process affecting the group. Nasal swabs should only be used to identify upper respiratory tract viruses. Deep nasopharyngeal swabs, positive for M. haemolytica and M. bovis, have been demonstrated to be representative of isolates present in the lungs. Samples obtained from transtracheal wash and/or bronchoalveolar lavage (BAL) can be used for virology, bacteriology, cytology and parasitology. However, the presence of bacterial isolates in nasopharyngeal swabs or tracheal and/or bronchoalveolar lavage needs to be interpreted with caution, in light of recent studies in which 63% of healthy calves were culture positive for bovine bacterial pathogens from BAL fluid. In one study, a high level of pathogens was found in the lungs of calves on arrival at feedlots. However, in another, the lungs were virtually sterile at the time of slaughter. Bacteria may be more likely to frequent the lung during high stress periods due to impairment of the mucociliary escalator mechanism.

Postmortem examination of untreated animals in the early stages of calf pneumonia can be useful, whereas repeatedly treated animals with chronic pneumonia are usually of little diagnostic value. Faeces should be examined for lungworm larvae, even though false negatives may occur if the animals are sampled before adult lungworm become patent.

Serum anti-BCV IgG antibodies were measured by ELISA in 195 samples collected from birth through weaning for 39 calves from Herd 2 that were involved in the mass treatment for BRD on August 12, 2016 in order to determine the mean antibody abundance and range at each sample acquisition time (Fig. 4). The mean (± standard deviation) anti-BCV antibody abundance declined from a maximum of 1186 ± 699 at birth to a low of 138 ± 88 at the time of mass treatment. Mean antibody abundance increased slightly following mass treatment, with mean antibody abundances of 176 ± 83 and 182 ± 95 at preconditioning and weaning, respectively.

Neutralizing antibody titers were measured in 60 of these samples from 12 randomly selected calves to determine the relationship between total anti-BCV reactive antibodies measured by ELISA and neutralizing antibody titers measured by a virus neutralization test (VNT). The effect of altering the strain of the test virus used in the VNT was also evaluated. A high positive mean correlation was observed between the ELISA and VNT assays regardless of the test virus used (Pearson’s rank correlation, ρ = 0.81 with BRCV_2014 strain and ρ = 0.91 with Mebus strain), indicating good to excellent agreement between the two tests under these conditions (Additional file 2). Thus, the ELISA was used for subsequent measurements of anti-BCV antibodies.

Number of herds that were antibody positive in the pooled milk samples was for BCV 46 (58.2%) and for BRSV 62 (78.5%). Ten herds were classified as NEG and 69 herds as POS The proportion of antibody positive herds did not differ significantly between the two areas under study for both BCV and BRSV (table 2). Geographical locations of the herds stratified by antibody status are shown in figure 1.

DNA extracted from pooled nasal swab medium was sent to the University of Nebraska-Lincoln Veterinary Diagnostic Center for bacterial diagnostics by multiplex qPCR using the QuantiFast multiplex PCR kit (Qiagen Inc.) and primers and probes designed to detect M. haemolytica, P. multocida, H. somni and Mycoplasma bovis. This test has been validated for use with bovine nasal swabs and lung tissue matrices.

The presence of antibodies to BRSV was tested by virus neutralization test (VNT). Serum samples were thawed, inactivated in water bath at 56 °C for 30 min, and diluted in duplicates from 1:2 to 1:1024 in 96-well microplates with 50 μL of 200 TCID50 BRSV suspended in Eagle’s minimum essential medium (DIFCO E-MEM®). The viral strain was previously titrated. Following incubation at 37 °C in 5% CO2 atmosphere for 1 h, 50 μL of Madin & Darby Bovine Kidney (MDBK) cells suspension, in E-MEM and 10% bovine fetal serum solution were added to the wells. Then, the plates were re-incubated in similar conditions for 96 h. Each test included a back titration and cell culture control. Samples were positive when cytopathic effect was inhibited at 1:2 dilution, and the titration was expressed as the inverse of the dilution, as geometric mean.

BK carried out bacteriological examination of samples, isolation and cloning of mycoplasmas and drafted the manuscript.

NFF carried out identification of mycoplasma isolates by DGI test and epi-immunofluorescence.

PA carried out molecular identification of mycoplasma isolates.

All authors read and approved the final manuscript.

Cases of calf pneumonia may not be detected by the animal keeper, but are more likely to be missed than misdiagnosed, as Sivula et al. have shown that keeper diagnosis is only 56% sensitive but 100% specific.

Early signs of calf pneumonia include elevated respiratory rate, fever, serous nasal discharge and at the most mild depression or inappetence. Since early treatment is the most important factor that prevents treatment failure, recognition at this stage would be preferable. The feasibility of daily measurement of the body temperature in high-risk periods is highly dependent on the housing system and handling facilities. When treatment is based on rectal temperature, thresholds of 40-40.3°C for feedlot cattle and 39.7°C for calves have been suggested. If measurement of the body temperature is not practical, early recognition and the success of treatment relies on good observational skills of the animal keeper. Evaluating calves for treatment using a screening system, such as the calf respiratory scoring chart developed at the University of Wisconsin, which is based on rectal temperature, character of nasal discharge, eye or ear appearance and presence of coughing, has been recommended for dairy calves. Apley suggests that treatment should be instituted on recognition of depression with undifferentiated fever, with depression being the more important of these two parameters.

Serum, nasal swabs and broncheoalveolar lavage (BAL) samples were collected from seven animals in the early phase of the disease, 2 days after the onset of the outbreak. Two weeks later, a second serum sample was collected from each of the animals except one that had died. The age at the first sampling was on average 143 days.

An indirect ELISA detecting serum antibodies to BRSV, BPIV3 and BCoV (SVANOVIR® BRSV-Ab, PIV3-Ab and BCV-Ab, Svanova Biotech AB, Uppsala, Sweden) was used, following the manufacturer’s instructions. In brief, the optical density (OD) reading of 450 nm was corrected by the subtraction of OD for the negative control antigen, and percent positivity (PP) was calculated as (corrected OD/positive control corrected OD) × 100. A sample was considered positive for antibodies if PP ≥ 10 and negative if PP < 10. The animals were regarded as acutely infected with BRSV, BCoV or BPIV3 if the animals seroconverted, i.e. if the first sample was negative and the second sample was positive or if they had a strong increase in antibodies, i.e. if the PP increased at least 70 %.

Real time polymerase chain reaction (RTqPCR) for detection of the N-protein of BRSV in the nasal swabs and BAL samples was performed as previously described by Klem et al..

Five of the six bulls with paired serum samples either seroconverted (four) or had a strong increase in BRSV antibody level in percent positivity of at least 70 % (one). Two of the six bulls seroconverted for BCoV and two of them had a strong increase in antibody level for BPIV3. The seven animals tested were positive for BRSV on nasal swabs (five) and/or BAL samples (seven) by RTqPCR (Table 1).

Post-mortem examinations were performed on four euthanized animals. Before euthanasia, the animals were in the terminal face of respiratory disease with anorexia, grunting with open mouth breathing, froth and stretched neck. Gross pathological and histological findings were recorded. For the histological investigation, sections of the lung tissue were stained with haematoxylin-eosin. Immunohistochemistry (IHC) was accomplished by use of a cryostat on frozen sections of lung tissue with anti-BRSV isotype IgG1 kappa (Nordic BioSite, Sweden) at a dilution of 1:100. In brief, the section was fixed in 10 % buffered formalin for 2 min followed by 2 min in 70 % alcohol. Inhibition was carried out with 0.05 % phenyl hydrazine in phosphate buffered saline at 37 °C for 40 min. The blocking was accomplished with N-serum from goat 1:50 in a solution of 5 % bovine serum albumin in tris-buffered saline for 20 min. Primary antibodies were diluted in 1 % bovine serum albumin in tris-buffered saline and incubated for 1 hour at room temperature (around 21 °C). The secondary antigen (Labelled polymer, HRP anti mouse) was incubated for 30 min before developing with 3-amino-9-ethylcarbazole for 20 min and counterstained with haematoxylin for 30 s. Sections stained by IHC were examined microscopically to determine the types of cells labelled for viral antigen. Lung tissue from all four animals examined for the presence of BRSV antigen with IHC was positive.

All the examined animals had a bronchointerstitial pneumonia characteristic of a BRSV-infection: the cranioventral parts of the lungs were consolidated and mucopurulent exudate was observed in the bronchus and small bronchi. The caudodorsal parts of the lungs were distended. There was emphysema, especially in the border between the consolidated cranioventral parts of the lung and the more normal caudodorsal parts. The tracheobronchial and mediastinal lymph nodes were markedly enlarged with a mottled cut surface. Microscopic lesions consisted of necrosis of the epithelium in the bronchioli and alveoli with rejection of epithelia to the lumen. Infiltration of neutrophil granulocytes and macrophages was also present in the lumen. There was hypermia and haemorrhage from capillaries into the bronchioli and alveoli. In the bronchioli, syncytial cells typical of the paramyxovirus were formed by fusion of epithelial cells and in the wall of the alveoli hyaline membranes were present.

By culturing of lung tissue from the four autopsied calves, Mannheimia haemolytica was identified in one, Pasteurella spp. in the second, and in the two remaining, no bacteria were detected.

Based on the antibody detection of paired serum samples from the six bulls, BRSV detection by PCR of the same animals, post mortem examination of four other animals and detection of BRSV by IHC of lung tissue from these four, the conclusion was that BRSV was the principal pathogen involved.

Proper specimen collection and delivery to a diagnostic lab is commonly neglected, and significantly impacts the diagnostic outcome. Antemortem samples for diagnostic testing should minimally include feces from acutely diarrheic animals prior to therapy with optional blood samples. Necropsy specimens from freshly sacrificed, moribund, or euthanized calves are of great value for diagnosis during severe outbreaks. Fresh and formalin-fixed gastrointestinal tissues (abomasum, small intestine, or colon) including ones from regional lymph nodes and liver should be collected along with colonic contents. Fresh fecal samples should be directly recovered from diarrheic animal into a specimen container with either rectal swabs or by rectal stimulation while avoiding environmental contamination (by soil, urine, or other feces). Once collected, the sample should be stored in a transporting medium or special stool container with refrigeration to maintain pathogen viability and sample integrity (e.g., reduced overgrowth of undesired bacteria and prevention of nucleic acid degradation). Samples of anaerobic bacteria (e.g., C. perfringens) should be kept in an oxygen-free transport medium during shipping if possible.

In order to select predictor variables to include in the model, univariable logistic regression analysis was performed. Only variables with a p-value lower than 0.2 for any of the outcome variables were included in the data analysis (Table 2). Variables that were not associated with dependent variables in a p-value of ≤0.2 and not included in the model were 'Mycoplasma bovis prevalence in cows', 'Mycoplasma bovis prevalence in heifers', 'relocating animals between the barns', 'frequency of overgrouping animals in the farm', 'using bull to inseminate cows', 'grazing youngstock', 'grazing cows' and 'number of livestock units within the farm'. Collinearity among all the outcome and explanatory variables was checked with the chi-square test. Variables 'housing of cows' (tied/loose) and 'barn type' (cold/warm) were highly collinear and had the same explanation so only the former was included in the models having better explaining capacity. Two herds were excluded from the data analysis owing to a large number of missing values. In order to avoid a reduction in the number of herds in the statistical analysis because of the absence of single values in some of the predictor variables, the original dataset was completed using an imputation technique in Stata 11 software (Stata Corporation, Texas, USA). The missing values were replaced using linear regression for multiple imputation for continuous variables and using logistic regression for binary variables (command mi impute). For each missing data point, five imputation values were generated and the mean was calculated.

Imputed values were created for four herds for the variable "BHV-1 prevalence in heifers" (4%), for six herds for the variables "BVDV prevalence" and "Mycoplasma bovis prevalence in heifers" (6%), for eight herds for the variable "BRSV prevalence in heifers" (8%), and for one herd for "Mycoplasma bovis prevalence in cows" (1%) in the initial dataset. In Model I, values were missing for some outcome variables for one herd and this herd was excluded from the data analysis. In Model II 24 herds were excluded from the analysis owing to missing values in either of the two outcome variables.

Multiple correspondence analysis (MCA) was used to obtain an overall view of the associations among variable categories in Model I, and to avoid problems arising due to multicollinearity. The test values are considered the standardized coordinates, and are used to interpret the significant variable categories to build each component, i.e. with absolute test values higher than the threshold value of 1.96. The test values are interpreted as the number of standard deviations from the centre of gravity of the analysis. The MCA was performed using XLSTAT (Version 2010.4.01; Addinsoft).

Logistic regression models were built to quantify estimates of the relationships among outcome and predictor variables. All the variables with p-value < 0.2 selected in the univariable logistic regression analysis were included in the multivariable logistic regression model. For the final multivariable model variables with p-value > 0.05 were excluded with backward elimination procedure. The logit function of Stata 11 (Stata Corporation, Texas, USA) was applied for logistic regression analysis. The change in regression coefficients was noted to identify important confounding factors (change in regression coefficients between the crude and adjusted value of > 20%), and biologically meaningful interaction were tested. The fit of the model was evaluated with the Hosmer-Lemeshow goodness-of-fit test. There was no indication of lack of fit in the logistic regression models.

Bovine kidney cells (MDBK/NBL-1; ATCC® CCL-22™) were cultured at 37 °C with 5% CO2, in DMEM (Fisher Scientific, Loughborough, UK) supplemented with 8% horse serum. Virus isolation and determination of the median of tissue culture infective dose were performed on MDBK cells. Virus isolation was performed as follows: The three nasal swabs, positive for BPIV3, were filtrated through a 0.22 μm filter (Millipore, Milford, MA, USA) and inoculated to a monolayer culture of MDBK cells cultured in Dulbecco’s modified eagle medium (DMEM, Fisher Scientific, Loughborough, UK) supplemented with 8% horse serum (Fisher Scientific, Loughborough, UK). MDBK cells were maintained at 37 °C in an atmosphere of 5% CO2. The cytopathic effect (CPE) was examined daily. The storage solution was exposed to a ten-fold dilution and filtered with a 0.22 μm filter. Then, the filtrate was inoculated into MDBK cells for 1 h. Finally, the culture medium was replaced with DMEM containing 2% horse serum. MDBK cells inoculated with filtrate were cultured in an incubator continually for 72 h. The propagation of the virus was performed three times.