The association of lung consolidation in beef × dairy cattle at weaning with feedlot growth performance, carcass characteristics, liver health, and liver microbiome diversity

Abstract

Bovine respiratory disease (BRD) is the costliest disease in the cattle industry and often compromises the immune system. The objective of this observational cohort study was to evaluate the impact of lung consolidation (LC) diagnosed at weaning (61 plus/minus 14 days of age) on feedlot growth performance, carcass characteristics, and liver health and microbiome in beef × dairy cattle. At 4 d post-weaning, LC was assessed by thoracic ultrasonography. The cattle (n = 139) either had ≥ 1 cm² LC in at least one lung lobe and were BRD positive (35 calves; BRD) or did not (< 1 cm²) and were negative (104 calves; CONTROL). Cattle were moved to the feedlot at 353 ± 53 d of age, where individual feed intake and body weights (BW) were recorded. Cattle were sent to slaughter when they reached a target final BW (steers = 680 kg and heifers = 635 kg). Liver scores and carcass data were collected. A subset (n = 29; 18 BRD cattle vs. 11 CONTROL cattle) had healthy liver tissue analyzed to investigate the association of LC at weaning with the liver microbiome diversity at slaughter. Only cattle with edible livers and no lung lesions were included in the microbiome analysis subset. Liver tissue samples were collected at slaughter and subsequently sequenced for microbiome analysis using an Illumina platform through targeted sequencing of the V4 region of the 16S rRNA gene. Mixed linear models were used to assess the effects of LC on growth performance and carcass characteristics with calf ranch, sex, and breed as fixed effects in the model. Generalized linear mixed models were used to assess the distributions of lung scores, liver scores, and quality grade at slaughter between LC and CONTROL cattle. To assess the effect of LC at weaning on the liver microbial communities at slaughter, the beta diversity (ADONIS) test was run, and the relative abundance of taxa is presented. There were no differences between BRD and CONTROL cattle for growth performance or most carcass traits (P > 0.05). However, the marbling score was greater (P = 0.05) in carcasses from CONTROL cattle (495 ± 7.82; LSM ± SEM) when compared with carcasses from BRD cattle (462 ± 13.84). The beta diversity in the liver did not differ (P > 0.05) between BRD and CONTROL cattle. Staphylococcus was the most abundant genus among the liver samples, regardless of health status at weaning. A diagnosis of BRD by LC in beef × dairy cattle at weaning (57 ± 14 d of age) reduced marbling and impacted quality grade.

Beef × dairy cattle affected with lung consolidation at weaning (approximately 2 months of age) have reduced marbling score at slaughter (approximately 16 months of age) compared to cattle without lung consolidation at weaning.

Introduction

From 1995 to 2015, the USDA estimated that 26.9% of the mortality in calves (beef and dairy) up to 227 kg was caused by complications associated with bovine respiratory disease (BRD; USDA-APHIS, 2017). These mortalities ultimately represent a loss of over $213 million to the cattle industry when considering the value of the calf alone. However, the financial losses related to BRD may be greater than 900 million US dollars (Peel, 2020) due to the costs of disease treatments (Dubrovsky et al., 2020), impacts on growth performance (Rhodes et al., 2021), and losses in the value of the calf itself (Peel, 2020). Beef × dairy calves are typically managed like traditional dairy calves during the pre-weaning and post-weaning period (Creutzinger et al., 2021), a strategy that exposes them to early-life stressors, such as early weaning, that can increase the risk of BRD when compared to beef cattle reared by the dam (Coetzee, 2013; McGill and Sacco, 2020). Market prices for day-old beef × dairy calves have been reported regularly over 1,000 dollars for an approximately 45 kg calf (USDA-AMS, 2025). In addition, these beef x dairy calves at 250 kg outpriced native (or full blood) beef calves at 250 kg in the spring of 2024 (USDA-AMS, 2025). Therefore, regardless of when these calves enter the beef supply chain, as day-olds or as feeder cattle at 250 kg, their value meets, or in some cases exceeds, that of beef animals. Given their growing market value (USDA-AMS, 2025) and demand in the beef industry, understanding the long-term impact of early-life BRD in these calves is not just a health priority but also an economic imperative. To the author’s knowledge, there are no studies that investigate how early-life respiratory disease influences the long-term performance of beef × dairy crossbred calves, thus allowing a unique study to offer novel insights into a population that is becoming increasingly important to the beef supply chain.

Bovine respiratory disease is an infection of lung tissue that causes cellular injury and lung lesions, also known as lung consolidation (Chai et al., 2022). Lung consolidation can be observed through thoracic ultrasonography, which has been adopted in research settings as the gold standard BRD diagnostic due to its high sensitivity rates (from 82% to 93%; Buczinski et al., 2018). Lung consolidation is associated with a reduction in average daily gain (ADG) up to 0.126 kg/d in pre-weaned dairy calves when compared to calves without lung consolidation (Cuevas-Gómez et al., 2021; Rhodes et al., 2021). During the feedlot receiving period (87 ± 3 d), Wilson et al., (2017) observed that beef calves that were treated for BRD, 1, 2, or 3 times before feedlot entry, entered the finishing period, 8, 39, and 64 kg lighter (P < 0.01) compared to calves that were not treated for BRD before feedlot entry. In addition, studies have shown that when cattle are treated for BRD in the feedlot, there is a reduction in hot carcass weight (HCW), ribeye area (REA), marbling, and 12th-rib fat thickness (RF), compared to cattle not treated for BRD in the feedlot (Wilson et al., 2017; Blakebrough-Hall et al., 2020). The impacts of this disease highlight the necessity of research to investigate the risks, impacts, and strategies to decrease BRD incidence at all stages of life throughout the beef system.

In addition to the impacts of BRD, liver abscesses in beef cattle also cause significant economic losses to the industry and are the leading cause of liver condemnation at slaughter (NBQA, 2022). The presence of severe liver abscesses in cattle can cause a reduction in ADG (Brown and Lawrence, 2010), HCW, REA, and RF (Grimes et al., 2024; Herrick et al., 2024). When accounting for fed- and cull-beef cattle slaughter, in 2022, the visceral losses due to liver abscesses were estimated at $59.9 million nationwide (Herrick et al., 2022).

While liver abscesses have been traditionally associated with ruminal acidosis and Fusobacterium necrophorum (Nagaraja and Chengappa, 1998; Amachawadi et al., 2017; Pinnell et al., 2023), many other agents could be involved in the etiology of the disease. For example, Trueperella pyogenes and Salmonella spp. have also been detected and isolated from liver abscesses (Amachawadi et al., 2017; Fuerniss et al., 2022; Pinnell et al., 2023). Bovine respiratory disease is also a complex disease where many factors and pathogens (bacteria and viruses) are involved, such as Mycoplasmopsis bovis, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and viruses such as Bovine Viral Diarrhea Virus, bovine herpesvirus 1, and many others (Klima et al., 2014). Other less commonly reported pathogens, like Fusobacterium spp. have also been identified in calf lungs (Raabis et al., 2021). Additionally, an increased relative abundance of Trueperella pyogenes in the nasal microbiome of steers with BRD has been suggested to be associated with BRD development (Centeno-Martinez et al., 2022). Alternatively, perhaps pathogens originating from the GI tract can disseminate to the liver and infect the lungs, such as Salmonella Dublin (Pecoraro et al., 2017). In addition, the presence of similar bacteria both in liver abscesses and the respiratory tract of cattle suggests a potential relationship between liver and lung health that could indicate pathways for pathogens, originating outside the rumen, to contribute to liver abscess formation. However, these hypotheses have not been fully explored.

While the lung microbial community of feedlot cattle infected with BRD pathogens has been investigated and demonstrated to differ from healthy cattle (Holman et al., 2015), the effects of early life BRD events on microbial diversity in the liver have not been explored. Given the significant financial losses and production impacts associated with BRD and liver abscesses, and the lack of lifelong studies on lung consolidation in beef × dairy cattle, the objectives of this study were to identify the lifetime effect of lung consolidation at weaning on the feedlot growth performance, carcass characteristics, liver health and microbial communities in beef × dairy cattle.

We hypothesized that beef × dairy cattle diagnosed with BRD by lung consolidation at 61 days of age (weaning) would have reduced feedlot growth performance, compromised carcass characteristics, and would have distinct beta diversity in the liver microbial communities when compared to cattle without lung consolidation at weaning.

Materials and Methods

This longitudinal observational cohort study occurred from 18 April 2023 to 12 September 2024. All animal care procedures were approved by the Penn State University Animal Care and Use Committee (IACUC: PROTO202001343). This study and manuscript were conducted following the quality standards of Strengthening the Reporting of Observational Studies in ­Epidemiology Veterinary Guidelines (Sargeant et al., 2016). A timeline demonstrating cattle transportation points and data collection timepoints is in Figure 1.

Figure 1.

Timeline of events and data collection for cattle enrolled (n = 139), showing key management events and sampling points from birth to slaughter.

Pre-weaning management

This study followed 139 spring-born beef × Holstein cattle (bulls, n = 68; heifers, n = 71) from birth to slaughter. The management protocols and feeding protocols for the pre-weaning and post-weaning phases are detailed in Fernandes et al. (2025). Briefly, 143 calves were sourced from two commercial dairy farms over a 53-d period and grouped into 3 consecutive groups based on birthdate (± 21 d). Calves had health exams at arrival, at day 28, at castration, 4 days after weaning, and at day 83 where they were assessed for joint swelling, umbilical infection, BRD, and diarrhea. Calf age within a group varied by no more than 14 days. Calves received colostrum and an ear tag with identification at the source dairy farm to ensure proper identification. Each source dairy farm had a herd of ∼1,500 lactating dairy cattle and was located within 40 miles from the calf ranches (n = 2) and the grower facility. Body weights (BWs) were taken on individual calves at arrival to the calf ranches at 1 to 4 d of age with a portable scale accurate to 0.1 kg (RM-CSmini, RoMech, Mini Crane Scale, Beijing, China), and at the calf ranches at 4 d post-weaning (weaning 61 ± 14 d), and at the grower facility (83 ± 21 d, and 238 ± 21 d; Fernandes et al., 2025) on a portable scale accurate to 0.1 kg (Model 640M, Avery Weigh-Tronix, Illinois Tool Works Inc, Glenview, IL, USA). The weights were taken at 4 d post-weaning, and again at 83 days to calculate post-weaning ADG (Fernandes et al., 2025), because this is a peak risk period for lung consolidation in calves, and lung consolidation has been associated with decreased temporal ADG in calves (Rhodes et al., 2021). Calves were individually housed and fed a total of 0.84 kg/day of milk solids, reconstituted to 6 L/d of milk replacer, and offered in two daily feedings for 49 d. A step-down weaning protocol was implemented starting at 49 ± 14 d of age, during which calves received 3 L/d of milk replacer once daily until weaning at 57 ± 14 d of age. Sire breeds represented in the study included Simmental (n = 42), Angus (n = 48), Red Angus (n = 22), and Charolais (n = 27). Of the 143 calves enrolled, 139 completed this study, which ended at slaughter at 480 ± 49 days of age.

Thoracic lung ultrasonography

Thoracic ultrasonography procedures occurred on all calves at 4 d post-weaning (61 ± 14 d), and the methodology is detailed in Fernandes et al. (2025). Lung ultrasonography was used to assess pulmonary health. Normal lung tissue was characterized by a continuous hyperechoic pleural line accompanied by reverberation artifacts. Lung consolidation was identified when a lobe appeared hypoechoic and the pleural line and reverberation artifacts were absent. The area of consolidation was estimated using 1-cm grid markers embedded on the ultrasound screen. A calf was classified as having lung consolidation if at least one lung lobe exhibited a consolidated area ≥ 1 cm². This threshold was selected based on previous research indicating that this level of lung consolidation is associated with a reduction in ADG of 0.11 kg/d through 50 d of age in dairy calves (Cramer and Ollivett, 2019). All cattle were vaccinated for bovine virus diarrhea (BVD) types 1 and 2, bovine rhinotracheitis (IBR) virus, parainfluenza3 (PI3) virus, and bovine respiratory syncytial virus (BRSV) with INFORCE 3 (Zoetis Inc., Kalamazoo, MI, USA) at 3 d of age and a second dose at 21 d (± 7 d), cattle also received vaccines for Mannheimia haemolytica, Clostridium chauvoei, septicum, novyi, sordellii, and perfringens Types C & D, at 97 ± 56 d with Alpha- 7 (Boehringer Ingelheim Animal Health USA inc., Duluth, GA, USA) and Bovi-Shield Gold 5 (Zoetis Inc., Kalamazoo, MI, USA). All vaccines were administered per labeling instructions.

At weaning, there were 35 of 139 (25%) calves that had lung consolidation ≥ 1 cm² in at least one lobe (BRD) and 104 of 139 (75%; < 1 cm²) calves that were negative for LC (CONTROL).

Post-weaning management

Calves were transported to a grower facility at 57 ± 6 d and commingled in a single bedded pen in a hoop barn. At the grower facility, cattle were fed a blended calf starter with free access of oat straw to approximately 140 d of age when they were transitioned to a corn silage-based total mixed ration ad libitum with free access to water during the whole period. Calves were implanted with Synovex Choice (100 mg trenbolone acetate, 14 mg estradiol benzoate; Zoetis Inc., Parsippany, NJ) at 262 ± 53 d of age and raised at the grower facility until 353 ± 53 d of age. The detailed management and diet during the post-weaning period are described by Fernandes et al. (2025).

Feedlot management

At 353 d of age, cattle were transported from the grower facility 38 km to the Pennsylvania Department of Agriculture Livestock Evaluation Center (LEC) in Pennsylvania Furnace, PA. The LEC feedlot consists of a gable-roof confinement barn with interior pens constructed of metal gates and cables on a concrete floor (3.3 m²/hd), open at the back of the pen to an exterior gravel lot (40.2 m²/hd). Cattle were stratified by weight, sex, and breed into three different pens and adapted to the feedlot and diet for 12 d. After the adaptation period, cattle were tagged with electronic identification tags (EID; Allflex Half Duplex, Merck Animal Health, Rahway, NJ), implanted with Synovex One Feedlot (200 mg trenbolone acetate, 28 mg estradiol benzoate; Zoetis Inc., Parsippany, NJ) and the final diet was initiated. Dietary ingredients included: corn silage, forage sorghum, dry rolled corn, dried distillers grains with solubles, soybean meal, limestone, and trace mineral salt with monensin. Individual feedstuffs were sampled approximately every week for the duration of the trial. Samples were used to determine the dry matter (DM) and adjust the inclusion of dietary ingredients. Additionally, every 14 d, 200 g of feedstuff samples were collected, dried at 55°C for 72 h, composited, and frozen at −20°C.

After the trial, composited feed samples were analyzed using wet chemistry methods for DM, neutral detergent fiber, acid detergent fiber, and crude protein by a commercial laboratory (Cumberland Valley Analytical Services, Waynesboro, PA). The diet contained (on a DM basis) 13.3% crude protein (50% soybean meal/50% dried distillers grain), 17% neutral detergent fiber, 7.7% acid detergent fiber, and 3.1 Mcal of metabolizable energy/kg. Individual animal feed disappearance was monitored daily using the GrowSafe Feed Intake Monitoring System (Model 4000E; Vytelle, LLC., Lenexa, KS) with 86% of the days included in the dataset. Cattle were fed in the system for ad libitum intakes, for example, feed always remained in the bunk.

Body weights and management procedures

Initial BW was determined by averaging the weights taken on two consecutive days, 12 d after arrival and after diet adaptation. Final BW was determined by averaging two consecutive weights taken the last 2 d before slaughter. Both initial and final BW were shrunk by 4% to account for gut fill because feed was always available. The targeted final BW for steers and heifers was 680 and 635 kg, respectively. However, due to facility restrictions, all remaining cattle were slaughtered by the last date regardless of BW. Cattle were sent to slaughter in five loads within 41 d. Cattle were transported 312 km to a commercial beef processing facility, located in Souderton, PA, and slaughtered according to the Humane Slaughter Act.

The ADG for the growing phase was calculated as the difference BW at feedlot entry and birth weight divided by d of age. The ADG for the feedlot phase was calculated as the difference between the feedlot final BW and feedlot initial BW divided by the number of d in the feedlot. Dry matter intake (DMI) was calculated as the means of as-fed intakes, monitored by Growsafe, multiplied by diet DM. Gain:feed (G:F) is reported as the ratio of ADG: DMI.

Carcass data collection

Cattle were slaughtered the day after transport to the packing plant. Individual animal ID was maintained through the harvest process to allow for identification. Carcass traits were recorded by multiple observers, but all observers were taught by a researcher with extensive experience in carcass trait research to ensure repeatability. Observers were blinded to individual cattle identification during the data collection at the packing plant.

Liver tissue sample. Livers considered inedible because of abnormalities were condemned by the USDA inspectors and removed from the line, where they were placed into a bag, and assessed on a sanitized, stainless-steel work surface to allow trained research personnel to score and collect tissue samples. Edible livers (normal) were removed from the line and placed in an adjacent stainless-steel cart to allow for tissue collection. Condemned livers were weighed using a stainless-steel scale provided by the facility, while edible livers were measured with a portable scale (ROYAL, EX315, Royal Ideas, China). All livers were weighed before sampling and with the gallbladder attached. Livers removed from the line were scored according to the Elanco liver scoring system (adapted; Greenfield, IN), as follows: edible = no abscess or abnormality, A− = 1 to 2 small abscesses (< 2.5 cm) or inactive scars, A = 1 or 2 large abscesses (≥ 2.5 cm) or multiple small abscesses (< 2.5 cm), A+ = multiple large abscesses (≥ 2.5 cm), A+adhesion (A+AD) = liver adhered to part of the gastrointestinal tract or diaphragm or both, A+open (A+OP) = open abscess, A+adhesion/open (A+AD/OP) = combination of A+AD and A+OP score and OA = other abnormalities, such as abnormal coloration, flukes, etc Livers were condemned by USDA inspectors from the line. One researcher inspected livers for abscesses, only one liver abscess was present in this study. Tissue samples were obtained from all livers using a sterile scalpel with the assistance of forceps. To avoid cross-contamination among condemned liver tissue collections, gloves, scalpels, and forceps were changed between liver samples collection. Healthy livers were sampled in line and knives were sterilized between samples of liver tissue. The cross-section of tissue samples was extracted from the central part of the liver only if the score was normal for this study. There was only one case of A+OP. Tissue samples were placed in sterile sampling bags (Nasco, Whirl-Pak, Filtration Group, USA) and transported in coolers with dry ice to the lab at the Animal Science Department at Pennsylvania State University and frozen at −80°C for further processing.

Lung data collection. Lungs were visually scored by trained research personnel at the offal table. This was done because lung lesions and scarring have been associated with liver abscesses at slaughter (Rezac et al., 2014), but there are no studies investigating the presence of LC at weaning (61 d of age) with the presence of LC, lesions, or scars at slaughter in beef × dairy cattle. Additionally, the lung score at slaughter was necessary to ensure that the CONTROL cattle enrolled in the liver microbiology analysis had healthy lung tissue (e.g., did not incur BRD later in their life). Two trained personnel with Fleiss kappa interobserver agreement κ  =  0.90 scored the lungs together on the first two dates and on alternate days thereafter. Lung score was recorded using a 4-point scale system modified by Magrin et al. (2021). A lung consolidation presented with a gray-red discoloration. Normal lung (score 0) = no visible consolidation or lesions associated with respiratory disease, slight (score 1) = one spot of 1 to 5 cm in diameter of lung consolidation, mild (score 2) = one spot of > 5 cm in diameter or several small spots, and severe (score 3) = lung consolidation area involving more than one lobe or signs of pleural adhesion to the thoracic cavity. The palpation of lung tissue was not used for scoring.

Carcass assessment. After slaughter, both sides of the carcass were weighed after removing the hide, head, organs, and kidney, pelvic, and heart fat (KPH) to determine HCW. The dressing percentage was determined by dividing HCW by the feedlot final BW. Three days after slaughter, one trained university researcher evaluated both sides of the chilled carcasses at the RF and REA and a different university researcher assessed the carcasses for marbling score. The average value of both sides was reported. In the event that only one side of the carcass could be assessed for any of the outcomes, then the singular value was reported from the side assessed. Yield grade (YG) was calculated using the USDA YG equation (USDA-AMS, 2017) with 2.5% estimated as KPH across all carcasses. Quality grade was defined by a combination of maturity and marbling as described by USDA-AMS (2017).

Liver microbial diversity enrollment criteria

Because the carcass order was not linked to individual animal ID until after slaughter, liver samples were collected from every animal, according to slaughter order. A subset of samples (n = 29; 18 BRD cases and 11 CONTROL) were selected to investigate the association of BRD diagnosed at 61 d with the liver microbiome relative abundance and beta diversity at slaughter. The participant flow diagram for the liver microbial analysis can be found in Figure 2. The livers enrolled in the comparison had to be graded as edible and from cattle that did not receive therapeutic antibiotics, and the corresponding ­animals’ lungs at slaughter had to be apparently normal (e.g., the absence of lobular and lobar lesions). Any animal with a lung lesion at slaughter was removed from the liver comparison. The CONTROL group for this analysis was healthy at each clinical exam throughout early life (farm arrival, 28 d, 4 d after weaning, and 83 d) described in Fernandes et al. (2025). All other parameters also had to hold true for the CONTROL livers enrolled. Samples were excluded if the liver was condemned and if samples had < 2,000 reads from the microbial DNA analysis.

Figure 2.

A participant flow diagram demonstrating enrollment and exclusion to follow-up for the association of BRD (lung consolidation ≥ 1 cm²) versus CONTROL (< 1 cm² lung consolidation) cattle at weaning with liver microbiome characteristics.

Liver samples DNA extraction and PCR amplification

The DNA extraction and PCR amplification were performed by Ganda Lab at the Pennsylvania State University (University Park, PA) adapted from the methodology described by Fonseca et al. (2024). Briefly, genomic DNA was isolated using the Kingfisher instrument with the MagMAX microbiome Ultra kit (Thermo Fisher Scientific, Austin, TX), according to the manufacturer’s instructions, and approximately 0.25 g of liver tissue originated from the samples were used. Both negative (DNA extraction kit reagents only) and positive (Zymobiomics microbial Community DNA Standard—Zymo Research Corporation, Irvine, CA) were included during DNA extraction to assess background contamination. The bacterial 16S rRNA V4 region was initially amplified from genomic DNA and the amplification was carried out for 30 cycles using the primer set comprised of 515F (5′-GTGYCAGCMGCCGCGGTAA- 3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) with respective positive and negative controls as described in ­Fonseca et al. (2024). The pooled and purified PCR products were sent to the Genomics Core Facility at the Pennsylvania State University (University Park, PA), subjected to library preparation, and sequenced on a 250 bp paired-end Illumina MiSeq platform (Illumina Inc., San Diego, CA).

Statistical analysis and bioinformatics

Based on previous research (Dubrovsky et al., 2019), we expected a BRD prevalence of ∼25% in this study. A post hoc power analysis was performed considering the observed variation in growth performance and carcass parameters such as ADG, DMI, G: F efficiency, marbling score, REA, HCW, dressing percentage, RF and YG. Given the final sample size, the study was powered at 80% to detect biologically significant differences, assuming a significance level of 0.05. All statistical procedures were performed in SAS (version 9.4, Cary, NC, USA). Significance was declared at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. When cattle arrived at the feedlot, they were stratified by BW, then allocated to pens balanced by, birthday cohort, pneumonia status at weaning, sex and breed. Mixed linear regression models (PROC MIXED) were used to assess the effects of lung consolidation at weaning (BRD or CONTROL, defined at LC severity ≥ 1 cm²) on feedlot growth performance (initial receiving BW and final BW in the feedlot, ADG, DMI, G:F, and age in days) and carcass characteristics (HCW, dressing percentage, REA, marbling score, RF, and YG). Lung consolidation at weaning (BRD or CONTROL), calf ranch, sex, and breed were used as fixed effects in the models. The least squares means were compared using pairwise differences.

Generalized linear mixed models (PROC GLIMMIX) was used to assess the association of lung consolidation at weaning (BRD or CONTROL) with the distribution of condemned livers and degree of lung scarring at slaughter. In addition, three generalized linear mixed models (PROC GLIMMIX) were generated to assess the association of lung consolidation at weaning (BRD or CONTROL) with the distribution of quality grades, separated as Select, Low Choice, or upper two-thirds Choice and above. Lung consolidation (BRD and CONTROL) at weaning was used as a fixed effect in the model. Data were run as a binomial distribution with a Satterthwaite adjustment. The least squares means were compared using pairwise differences with an inverse link function. An ordinal logistic regression model (PROC LOGISTIC) with logit link distribution was run to assess the odds of lung consolidation at weaning with Select quality grade adjusting for consolidation at weaning, breed, and sex as fixed effects.

The bioinformatics analysis was done using R (version 4.4.2, 2023) following a modified approach based on Fonseca et al. (2024). The Decontam package (Davis et al., 2018) was used to assess the background microbial signal and remove potential contaminants prior to further analysis. Contaminant detection was performed using the prevalence method with a stringent threshold of 0.5 and DNA extraction blanks as well as PCR no-template controls were included as negative controls to distinguish contaminants from true community members. Positive controls (ZymoBiomics Community Standard) were retained as biological samples to ensure expected taxa were not filtered out. To assess the effects of lung consolidation at weaning (BRD or CONTROL, defined at LC severity ≥ 1 cm²) with liver microbial community, statistical analyses were conducted on beta diversity (between-sample) and relative abundance of bacterial genera and phyla. For beta diversity analyses, bacterial community data were transformed to center log-ratio (CLR) and visualized in a principal coordinates analysis (PCA) at the genus level using the microViz package, version 0.12.6 (Barnett et al., 2021). Differences in beta diversity were analyzed with permutational multivariate analysis of variance (PERMANOVA) using the Adonis test from the vegan package with 999 permutations on Aitchison distances (Oksanen et al., 2022). Genus and phylum-level compositional bar plots were generated using tidyMicro. Complete code and Phyloseq are available at https://github.com/IngridLBFernandes/BRD-feedlot-growth.

Results and Discussion

Descriptive statistics

Of the 143 animals enrolled at weaning, 139 completed the trial. At the grower facility, three steers died, 2 because of lobar pneumonia in two lobes per confirmed necropsy by the herd veterinarian, and one for unknown causes (∼225 d of age). One heifer was injured during transportation to the feedlot, no treatment was effective in treating the injury, and this animal was euthanized and not part of the study. Animals that did not complete the trial were excluded from analysis.

Association of lung consolidation at weaning with feedlot growth performance

For this study, at the thoracic ultrasonography exam at 4 d post-weaning, 25% (35/139) of calves had lung consolidation ≥ 1 cm² in at least one lobe. Dubrovsky et al. (2019) reported a prevalence of 22.8% of BRD cases in a study involving 11,470 pre-weaned dairy calves (birth to 79 d of age) in California, which was similar to our study findings. The results in this study were consistent with Dubrovsky et al (2019) because beef × dairy calves are limited-fed milk and weaned by two months of age, like dairy calves, suggesting similar management conditions. Beef × dairy calves experience much higher rates of BRD status after weaning than pureblood beef calves raised by the dam and weaned at 7 months of life. For example, Dewell et al. (2006) and Hanzlicek et al. (2013) reported much lower BRD prevalence in pre-weaned beef calves (5% and 3%, respectively). It is difficult to make comparisons since beef producers wean beef calves much later than beef × dairy calves and feed them differently. However, beef × dairy calves are developing immunocompetence when milk is removed, likely making them more susceptible to the disease (Cortese, 2009). These data relate to the etiology of the disease, where stress and environmental factors are risk factors (Madureira Ferreira et al., 2024).

Regardless of LC at weaning, CONTROL and BRD cattle had similar feedlot growth performance, reported as initial and final BW, ADG, DMI, and G:F (P > 0.05; Table 1).

Table 1.

Impact of lung consolidation diagnosed at weaning (61 ± 14 d of age), on feedlot growth performance of beef × dairy cattle

Item	CONTROL1	BRD1	SEM	P-value
n, head	104	35	–	–
Growing phase2
ADG, kg/d3	1.10	1.10	0.01	0.76
Feedlot2
Initial BW, kg	432	428	5.90	0.48
Final BW, kg	641	638	6.11	0.69
ADG, kg/d3	1.75	1.77	0.04	0.73
DMI, kg/d4	13.82	13.83	0.23	0.96
G: F5	0.126	0.128	0.003	0.58
Days of age, d6	477	474	2.50	0.30

¹Diagnosed at weaning by thoracic ultrasonography. CONTROL: negative thoracic ultrasonography or < 1 cm² of lung consolidation. BRD: ≥ 1 cm² of lung consolidation in at least one lobe.

²Growing phase from birth to feedlot entry at 353 ± 53 d of age. Feedlot phase from 353 ± 53 d to 474 ± 54 d of age.

³Average daily gain = (final BW—arrival BW)/days on feed in the feedlot.

⁴Dry matter intake.

⁵Efficiency gain: feed = ADG/DMI.

⁶Days of age from birth to slaughter.

Fernandes et al. (2025) divided calves into three periods; period 1 (birth to 61 ± 14 d of age), period 2 (61 to 83 ± 21 d of age), and period 3 (83 to 238 ± 21 d of age), to identify how lung consolidation at weaning affects growth performance for short periods. The authors reported that animals with lung consolidation had decreased ADG (−0.14 ± 0.03 kg/d; P < 0.01) for 2 wk post-weaning. In that study, 2 wk post-weaning represented 83 d of age; it is important to highlight that calves undergo adipogenesis at this age, depositing fat cells in the muscle, which can impact carcass quality later in the animal’s life. However, calves compensated for that loss over time, with no differences (P > 0.05) in ADG by d 238 among calves that had been diagnosed at weaning with and without lung consolidation. The calves fed by Fernandes et al. (2025) were the same cattle used in this study. Therefore, when calculating the overall growing phase ADG from birth to feedlot entry, no differences were observed between CONTROL and BRD calves (P = 0.76; Table 1). During the disease process, the immune system will respond, causing inflammation, modifying metabolism, and decreasing DMI, which might have negative consequences on growth performance (Krehbiel, 2020). Therefore, with disease resolution, cattle are expected to return to their basal homeostasis as the febrile response is no longer active (Krehbiel, 2020). Our data suggest that young calves diagnosed with BRD at 57 ± 14 d of age have an innate ability to overcome the disease insult over time. However, the beef × dairy production system differs greatly from conventional beef cattle production.

Schneider et al. (2009) reported a more pronounced drop in ADG (0.37 ± 0.03 kg) when cattle treated for BRD were compared to healthy cattle over the course of the feedlot diet acclimation period. Cattle in that study averaged 288 ± 44 kg and 287 ± 60 d of age and were, thus, much older than the cattle diagnosed in our study. Schneider et al. (2009) reported that feedlot ADG in cattle treated for BRD was reduced to a greater extent (0.37 ± 0.03 kg) during the feedlot acclimation period compared to the overall reduction in ADG (0.07 ± 0.01 kg) observed across the entire feeding period when compared to healthy cattle. Results from these studies suggest that while a complete ability to overcome disease insult was not possible, the insult was lessened due to compensatory gain after feedlot acclimation. Because the cattle from the aforementioned study were sourced from 10 different feedlots across the Midwest and Southeast of the United States, it is important to note that there were no vaccination, health, or treatment records prior to the feedlot entry reported whereas all cattle in our study had similar health management. Wilson et al. (2017) reported slightly conflicting results to Schneider et al. (2009) in feedlot growth performance of cattle when BRD was diagnosed at the feedlot receiving period averaging 217 ± 20 kg. The authors observed an increase in overall ADG at the end of the study with increasing BRD treatments during the receiving period (0X, 1X, 2X, and 3X or 4X), suggested that cattle overcame the ADG insults during the receiving period, which conflicts with findings from Blakebrough-Hall et al. (2020), that reported a linear decrease in ADG (1.8, 1.7, 1.3, and 1.1 kg/d) during the feedlot with the increasing incidence of BRD treatment (0X, 1X, 2X, and ≥ 3X) was reported.

There is a challenge in comparing compensatory gain after BRD in early life in this study vs. studies that evaluated BRD in the feedlot acclimation period because of the differences in age, environment, and diet. It is also important to note that the definition for BRD used in this study is the smallest amount of lung consolidation (e.g., LC = 1 cm²) that has been observed to negatively affect calf growth (Cramer and Ollivett, 2019); which may suppress the innate immune system, but with time, the adaptive immune system can build a response, allowing the animals to fully recover (Ackermann et al., 2010). For example, dairy heifers with more advanced lung consolidation in the pre-weaning period (e.g., LC = 3 cm²) produced 525 kg less milk in the first lactation when compared to controls, suggesting that early-life health events impact future cattle outcomes (Dunn et al., 2018). Moreover, two of the conventional feedlot studies compared here (Schneider et al., 2009; Wilson et al, 2017) hint at a compensatory effect; however, DOF was only reported by Wilson et al. (2017) and those cattle had less than 200 DOF from feedlot entry to harvest. Blakebrough-Hall et al. (2020) reported a maximum of 120 DOF in their study, cattle were not able to compensate for the gain lost due to BRD insult. Schneider et al. (2009) did not report time on feed. We suggest, therefore, that the ability or inability of the cattle to overcome or compensate for BRD insult may be related to disease severity, the number of cases of BRD, age at diagnosis, and, consequently, how much time cattle are fed after the insult. Despite the body of literature on dairy calves finished for beef production, most studies begin at feedlot entry. To the authors’ knowledge, no studies have investigated the impacts of untreated early calfhood respiratory disease events on the long-term growth performance of cattle entering the beef industry, suggesting this is the first for beef × dairy cattle.

Association of lung consolidation at weaning with carcass characteristics at slaughter

Hot carcass weight, dressing percentage, REA, RF, and YG were similar (P > 0.05) among CONTROL and BRD cattle (Table 2). In this study, the marbling score was reduced (P = 0.05) by 6.5% in BRD cattle when compared to CONTROL cattle. Cattle with LC at weaning tended (P = 0.08) to have a greater percentage of carcasses that graded Select (20.0%) and had (P = 0.04) a reduced percentage of carcasses that graded upper two-thirds Choice and Prime (14.3%) when compared to CONTROL cattle (8.7 and 33.7%, respectively; Table 2). Therefore, cattle with lung consolidation at weaning tended to have 3.05 times greater odds of having a poorer carcass quality grade, select vs. prime/choice, than cattle that were healthy at weaning (95% CI 0.93 to 10.02; P = 0.07). Most beef cattle studies have investigated the effects of BRD observed during the feedlot on carcass traits (Garcia et al., 2010; Blakebrough-Hall et al., 2020). For example, Schneider et al. (2009) observed, through treatment records and lung scores at slaughter, that cattle with BRD in the feedlot decreased HCW by 2.5%, RF by 1.5%, and marbling score by 2.5% when compared to healthy cattle. Feedlot cattle with inactive and active lung lesions at slaughter yielded 55 and 125% more carcasses that graded Standard, respectively, when compared to cattle without lung lesions(Gardner et al., 1999). While this report demonstrates the impact of lung lesions observed at time of slaughter on carcass quality, overwhelmingly there is a dearth of information regarding the long-term effects of early-life diseases on cattle growth and production.

Table 2.

Impact of lung consolidation diagnosed at weaning (61 ± 14 d of age), on carcass characteristics of beef × dairy cattle.

Item	CONTROL1	BRD1	SEM	P-value
n, head	104	35	–	–
HCW, kg	394	394	3.86	0.92
Dressing Percentage, %	61.5	61.7	0.28	0.53
Ribeye area, cm2	97.39	96.58	1.24	0.56
Marbling score2	495a	463b	14.23	0.05
12th rib fat thickness, cm	1.15	1.11	0.06	0.56
Yield Grade3	2.11	2.11	0.10	0.97
Quality grade3, % (n)
Select	8.7 (9/104)	20.0 (7/35)	6.8	0.08
Low Choice	57.7 (60/104)	65.7 (23/35)	8.0	0.41
Upper 2/3 Choice and above	33.7 (35/104)	14.3 (5/35)	5.9	0.04

¹Diagnosed at weaning by thoracic ultrasonography. CONTROL: negative thoracic ultrasonography or <1 cm² of lung consolidation. BRD: ≥ 1 cm² of lung consolidation in at least one lobe.

²300 = Slight⁰⁰, 400 = Small⁰⁰, 500 = Modest⁰⁰, 600 = Moderate⁰⁰ (U.S. Department of Agriculture, 2017).

³Yield and quality grade were calculated as described by USDA (1997).

The reduction in marbling score observed in BRD cattle in our study may be attributed to the fact that one of the most critical periods for intramuscular adipogenesis has been suggested to occur between the late fetal stages up to approximately 250 d of age in beef cattle (Du, 2023). Hyperplasia, which is the increase in the number of intramuscular adipocytes, is still increasing during this period while after 250 d of age, adipocyte hypertrophy, which is the increase in the volume of the adipocytes present in the muscle, replaces hyperplasia (Du, 2023). In conventional beef cattle operations, calves remain with the dam for 180 to 200 days of life. Thus, most attempts to modify marbling in this window have been conducted in early weaning systems (90 d of age +) through grain programs (i.e., creep feeding; Shike et al., 2007). Our data would suggest that perhaps adipogenesis must be manipulated earlier. In our study, the growth insult from BRD diagnosed at weaning was prevalent between 60 and 83 d of age (Fernandes et al., 2025). We hypothesize that BRD at weaning reduced the number of intramuscular adipocytes and that this impact was observed as reduced marbling in the carcass at slaughter, but future work should investigate if this is the case.

These findings raise concerns for the beef industry considering that over 70% of producers sell cattle in systems where the quality grade is part of the cattle price calculation (USDA, 2025). Blakebrough-Hall et al. (2020) reported a reduction of 7% of the total slaughter value when cattle treated twice for BRD were compared to cattle that were not treated in the feedlot. While interesting, the aforementioned trial involved Bos indicus cross cattle in an Australian feedlot. Additional economic studies need to be evaluated in the U.S. to investigate the long-term financial losses of early-life health events on cattle targeting the beef industry. The segregation of the cattle industry makes these studies complex. In our study, cattle moved as they would in a dairy system targeted for beef production: from the dairy to a wet calf ranch, to a grower, to the feedlot. Thus, the stressors associated with moving cattle through four operations, as is common, were mimicked in our study.

Association of lung consolidation at weaning with liver condemnation, lung consolidation and liver microbiota at slaughter

There was a total of 12 condemned livers at slaughter. Out of the 12 condemned livers, 3 were from BRD cattle and 9 from CONTROL cattle. Across the entire study population, 9% (12/139) of the cattle slaughtered had condemned livers (Table 3). A similar percentage of liver condemnation at slaughter (8.6 and 8.7%, respectively) were observed for BRD and CONTROL cattle. The incidence of liver condemnation in this study is contrary to the previously reported number of 50.18% of liver abscesses in beef × dairy cattle (Grimes et al., 2024), but in agreement with the observations that cattle fed in the Northeastern U.S. often have low incidence of liver abscesses (Herrick et al., 2022). Contrary to our hypothesis, there was no association of BRD at weaning with liver condemnation (P = 0.99) or lung lesions at slaughter (P = 0.27; Table 3). We assessed the association of lung consolidation at weaning with liver microbial diversity and abundance because Raabis et al. (2021) had sourced samples from the nasopharynx of dairy calves that were positive for Fusobacteria, a pathogen phylum previously isolated and detected from liver abscesses (Amachawadi et al., 2017; Fuerniss et al., 2022). Thus, we wanted to explore the relationship between liver microbial diversity and BRD, observed at weaning to determine if there were carryover effects of early life disease on the microbial diversity of the liver, an organ that is often compromised in beef production systems.

Table 3.

Distribution of liver abnormalities and lung score at slaughter in beef × dairy cattle diagnosed with lung consolidation at weaning (61 ± 14 d of age)

Item	CONTROL1	BRD1	SEM	P-value
Edible liver, % (n/total)	91.4 (95/104)	91.4 (32/35)	4.7	0.99
Condemned liver score, % (n/total)2	8.7 (9/104)	8.6 (3/35)	4.7	0.99
A-	4.8 (5/104)	2.9 (1/35)	2.8	0.63
A	2.8 (3/104)	0	1.6	0.98
A+	0	0	–	–
A+OP	1 (1/104)	0	1.0	0.98
Other	0	5.7 (2/35)	3.9	0.97
Lungs scored > 1, % (n/total)	49 (51/104)	60 (21/35)	0.40	0.27
Lung score at slaughter, % (n/total)3
Score 0 (healthy)	51 (53/104)	40 (14/35)	8.3	0.27
Score 1 (mild)	30.8 (32/104)	34.3 (12/35)	8.0	0.70
Score 2 (moderate)	11.5 (12/104)	14.3 (5/35)	5.9	0.67
Score 3 (severe)	6.7 (7/104)	11.4 (4/35)	5.4	0.38

¹Diagnosed at weaning by thoracic ultrasonography. CONTROL: negative thoracic ultrasonography or < 1 cm² of lung consolidation. BRD: ≥ 1 cm² of lung consolidation in at least one lobe.

²Liver condemned at slaughter by a USDA inspector. Liver abnormalities observed by university personnel included: A– = liver with 1 or 2 small abscesses (< 2.5 cm) or inactive scars; A = liver with one or two small abscesses, or up to 4 grouped abscesses under 2.5 cm diameter; A+ = liver with one large abscess (> 4 cm) or multiple abscesses (> 2.5 cm); A+OP = liver with ruptured abscess; other = steatosis, flukes and bruise.

³Lung scored for consolidations at slaughter by university personnel. Included all severity of consolidation (0 = lung without consolidation; 1 = consolidation of lung lobes affecting more than 5% to 15% of lung tissue; 2 = pleural adhesions and consolidation affecting more than 15% to 50% of lung tissue; 3 = pleural adhesions and consolidation affecting more than 50% of lung tissue.).

The beta diversity of healthy livers from BRD (n = 18) and CONTROL (n = 11) cattle did not differ (P = 0.47; Figure 3). To the author’s knowledge, no studies have evaluated the microbial community in healthy livers sequenced from beef × dairy cattle using the 16S rRNA technique. This gap likely exists because liver tissue is a low-biomass sample, meaning it contains small amounts of microbial DNA, which makes it technically challenging to analyze using traditional sequencing methods. As a result, failed or incomplete sequencing is a common and expected limitation when working with this type of tissue. Despite these challenges, it is still unclear what the common microbiota in healthy livers is and what changes to expect. Therefore, this study begins to establish foundational knowledge in this area.

Figure 3.

Beta diversity comparisons from livers collected at slaughter among beef × dairy cattle (n = 29) without (CONTROL) or with (BRD) lung consolidation (≥ 1 cm² in at least one lobe) at weaning (61 ± 14 d of age) at the genus level, analyzed using the Adonis test with 999 permutations on Aitchison distances.

When cattle are infected with BRD, the immune system initiates a response against the infection, creating an inflammatory reaction (Gifford et al., 2012). The inflammation can incite the febrile response, muscle catabolism, changes in liver protein synthesis, and alterations to carbohydrate and lipid metabolism (Gifford et al., 2012). The lipopolysaccharides present in gram-negative bacteria such as Pasteurella multocida and Mannheimia haemolytica (BRD pathogens) will trigger an immune reaction that causes a decrease in carbohydrate stores (Werling et al., 1996). In addition, feedlot cattle diagnosed with BRD by clinical signs were demonstrated to have a distinct microbial community in the nasopharynx and trachea compared to healthy cattle (Timsit et al., 2018). Based on previous work on these cattle, P. multocida was the most abundant microflora isolated from the nasopharyngeal swabs of cattle diagnosed with BRD by LC (Fernandes et al., 2025). Among these changes observed during BRD, we hypothesize that liver metabolism and changes in microbial diversity in the site of infection may be contributing factors to the changes in the liver microbial community.

The most abundant genera observed among liver samples from BRD cattle at weaning in this study were Staphylococcus (16.15 ± 0.29%) and Escherichia-Shigella (13.89 ± 0.23%; ­Figure 4). The most abundant genera observed among liver ­samples from CONTROL cattle were Staphylococcus (31.63 ± 0.38%) and Sphingomonas (11.70 ± 0.30%; Figure 4). In our study, 99% of the microbial community at the phylum level in the liver samples from BRD cattle at weaning were ­comprised of Proteobacteria (42.60 ± 0.35%), Firmicutes (27.45 ± 0.30%), Actinobacteriota (16.57 ± 0.27%), Verrucomicrobiota (5.90 ± 0.15%), Deinococcota (5.01 ± 0.10%) and Sumerlaeota (1.66 ± 0.07%; Figure 5). In contrast, the liver samples from CONTROL cattle had Firmicutes (35.10 ± 0.37%), Proteobacteria (32.58 ± 0.37%), Actinobacteriota (26.66 ± 0.34%), Deinococcota (3.86 ± 0.01%), and Bacteroidota (1.81 ± 0.06%) comprising 100% of the microbial community (Figure 5).

Figure 4.

Relative abundance of the most abundant taxa from livers collected at slaughter among beef × dairy (n = 29) without (CONTROL) or with (BRD) lung consolidation (≥ 1 cm² in at least one lobe) at weaning (61 ± 14 d of age) at the genus level. Each color represents one genus group.

Figure 5.

Relative abundance of the most abundant taxa from livers collected at slaughter among beef × dairy (n = 29) without (CONTROL) or with (BRD) lung consolidation (≥ 1 cm² in at least one lobe) at weaning (61 ± 14 d of age) at the phylum level. Each color represents one genus group.

While we observed that beta diversity was not associated with BRD at weaning, it appears that the Proteobacteria phylum is more abundant in cattle with a history of BRD at weaning compared to CONTROL, which had a greater abundance of the Firmicutes phylum. This agrees with Gaeta et al. (2017), where dairy calves with BRD at 28 d of life also had a greater proportion of Proteobacteria phylum compared to the controls detected from nasopharyngeal swabs. Our results are also supported by observations from Timsit et al. (2018), where there was a greater proportion of phylum Firmicutes identified from nasal and tracheal samples of healthy cattle and Proteobacteria were more abundant in feedlot cattle with BRD. While more research is needed, this current study provides initial evidence that early-life disease may influence the proportions of phyla present in the healthy liver of cattle later in life. These findings should be interpreted with caution because we only selected livers that had > 2,000 reads for comparisons. The low biomass present in liver is a study limitation, but as it is the first study to investigate effects of early life BRD on microbial beta diversity and relative abundance, we believe it is worth presenting the results as proof of concept. While the aforementioned studies agree with our findings about relative abundance, they differ because they sampled cattle at the time of clinical diagnosis of disease and sampled the site of infection. However, this is important because healthy cattle may present a more abundant phylum of Firmicutes, and cattle that experience an insult of BRD at weaning may continue to have a greater proportion of the Proteobacteria phylum in the liver throughout their lives. Future research should investigate if cattle with liver condemnation have different relative abundance and microbial diversity of the microflora from the liver compared to controls.

Conclusions

This study examined the impacts of lung consolidation at weaning on the growth performance, carcass traits, and liver health and microbial community of beef × dairy cattle at slaughter. In this study, marbling score, recorded at slaughter, was reduced in cattle diagnosed with BRD, 400 d after the diagnosis. These data demonstrate that the early life health management in beef × dairy calves is critical to produce quality carcasses. While others have reported that the ability to impact marbling score may last out to several hundred days of age in cattle, our results indicate that insults occurring much earlier, between 60 and 83 days of age, have lifelong impacts. While lung consolidation at weaning did not affect feedlot growth performance or other carcass traits, the reduction in marbling score impacts profitability for producers selling on a grid-based system. Additionally, cattle with lung consolidation at weaning did not have different liver microbial beta diversity at slaughter compared to cattle without lung consolidation at weaning. However, we observed that calves diagnosed with BRD at weaning had more relative abundance of the Proteobacteria phyla than the Firmicutes phyla within samples. Overall, these findings indicate that BRD, diagnosed at weaning (e.g., 2 months of life), compromised marbling development and affected the relative abundance of phyla in the liver.

Glossary

Abbreviations:

ADG

average daily gain

BRD

bovine respiratory disease

BW

body weight

CLR

center log ratio

DM

dry matter

DMI

dry matter intake

DNA

deoxyribonucleic acid

DOF

days on feed

G:F

gain to feed efficiency

HCW

hot carcass weight

hd

head

KPH

kidney, pelvic and heart fat

LC

lung ­consolidation

LEC

Pennsylvania Department of Agriculture Livestock Evaluation Center

PCA

principal coordinate analysis

PCR

polymerase chain reaction

REA

ribeye area

RF

12th-rib fat thickness

YG

yield grade

Author Contributions

Ingrid L.B. Fernandes (Data curation, Formal analysis, Methodology, Project administration, Visualization, Writing—original draft, Writing—review & editing), Melissa C. Cantor (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Visualization, Writing—review & editing), Ana Fonseca (Data curation, Formal analysis, Visualization, Writing—review & editing), and Erika Ganda (Conceptualization, Formal analysis, Methodology, Resources, Visualization, Writing—review & editing), Tara L Felix (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing—review & editing)

Conflict of interest statement. The authors have not stated any conflicts of interest.