Evidence of respiratory infection by bovine coronavirus in a neonatal calf

Abstract

Background

Bovine coronavirus (BCoV) is a major pneumoenteric pathogen that causes diarrhea in calves and respiratory disease in cattle of all ages. While BCoV-associated diarrhea is well-documented, reports of respiratory infection in neonatal calves remain limited, particularly in the Republic of Korea (ROK). Here, we describe a case of BCoV-induced pulmonary infection in a calf that presented without clinical respiratory signs.

Case presentation

A 7-day-old Holstein calf developed white, watery diarrhea and dehydration that subsequently progressed to astasia, showing no respiratory signs. Following euthanasia due to poor prognosis, necropsy revealed no gross lesions. However, real-time RT-PCR detected BCoV only in the lungs, whereas all other tissue samples, including the intestines, tested negative. Histopathologic examination revealed mild bronchiolitis and interstitial pneumonia, and immunohistochemistry confirmed the presence of viral antigen in bronchiolar epithelial cells and alveolar macrophages.

Conclusions

This case represents a naturally occurring BCoV pulmonary infection in a neonatal calf in the ROK. These findings indicate that BCoV can establish primary respiratory infection at a very early age without typical respiratory signs. Overall, this observation underscores the potential role of BCoV as an important contributor to respiratory disease in calves.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-026-05594-6.

Background

Bovine coronavirus (BCoV) is a pneumoenteric virus belonging to the species Betacoronavirus 1, together with human coronavirus OC43 and coronaviruses of other domestic and wild animals [1, 2]. BCoV is an economically important pathogen in the cattle industry, responsible for neonatal calf diarrhea, winter dysentery in adult cattle, and respiratory disease in cattle of all ages [3, 4]. Infected calves typically develop watery to hemorrhagic diarrhea, whereas respiratory infection can cause nasal discharge, cough, pyrexia, and pneumonia, and can result in substantial productivity losses in affected herds [5, 6].

BCoV shows tropism for both the gastrointestinal and respiratory tracts. Enteric BCoV, which is the most prevalent infection type, replicates in villous and crypt epithelial cells, leading to villous atrophy, malabsorption, and diarrhea [3]. By contrast, respiratory infection causes epithelial damage in the nasal turbinate, trachea, and lungs [7]. Since viruses isolated from either the intestinal or respiratory tract are antigenically and genetically indistinguishable, several studies indicate that enteric and respiratory BCoVs can replicate at either site. This overlap complicates assigning the infection origin and accurately assessing BCoV pathogenicity [8, 9].

Bovine respiratory disease complex (BRDC) comprises diverse clinical manifestations and pathological lesions affecting the respiratory tract of cattle [10] and is a leading cause of morbidity and economic loss in the cattle industry due to reduced growth performance, increased mortality, and treatment costs [11, 12]. BRDC has a multifactorial etiology involving complex interactions among viral and bacterial pathogens, environmental stressors, and host immune responses [13, 14]. Although several pathogens have been implicated in BRDC, BCoV has recently gained attention due to its frequent detection in the bovine respiratory tract [15–18]. BCoV has been detected in nasal secretions, tracheal or lung washes, and lung tissues from both clinically healthy and diseased cattle [4, 18–20]. In addition, several studies have demonstrated that BCoV infection can induce damage to the respiratory epithelium and may facilitate secondary bacterial infections [8, 21]. BCoV has been identified as an important contributor to BRDC in Japan [22, 23].

Nevertheless, in the Republic of Korea (ROK), research on BCoV has mainly focused on its role as an enteric pathogen, particularly causing diarrhea in young calves [9, 24–26]. Although BCoV has been implicated in respiratory disease, reports describing respiratory infection in cattle remain limited in the ROK [27]. In this case report, we describe a naturally occurring BCoV infection detected in the lung of a diarrheic calf without clinical respiratory signs.

Case presentation

In April 2025, a 7-day-old Holstein calf exhibited white, watery diarrhea for 2−3 days (Fig. 1) and subsequently developed astasia. The calf showed severe dehydration and anorexia. Because the prognosis was poor, the owner decided to euthanize the calf. A local veterinarian induced anesthesia with intramuscular administration of xylazine (Rompun^®, Elanco Animal Health, Greenfield, IN, USA; 0.03 mg/kg), performed euthanasia by intravenous administration of T-61 (MSD Animal Health, Rahway, NJ, USA; 0.1 mL/kg), and subsequently conducted a necropsy. No gross lesions were observed in the lungs (Fig. 2) or any other organs. Tissue samples were collected from multiple organs, including the tonsil, trachea, lungs, heart, liver, spleen, kidney, duodenum, jejunum, ileum, cecum, colon, and rectum. Fecal samples were not collected at necropsy. All samples were submitted immediately to the Animal Immunology Laboratory, Kyungpook National University, for further analysis.

Fig. 1.

White, watery diarrhea observed in this calf

Fig. 2.

Gross lesions in this calf. No macroscopic lesions were observed in the lungs. The lungs appeared grossly normal, with no evidence of consolidation or inflammatory exudates

Total RNA was extracted from all tissue samples with the RNeasy Mini Kit (Qiagen, Hilden, Germany). DNA was also extracted from the lung, small and large intestines with the DNeasy Blood & Tissue Kit (Qiagen). Real-time RT-PCR was performed using the One Step TB Green^® PrimeScript™ RT-PCR Kit (Takara, Shiga, Japan) to detect bovine viral diarrhea virus (BVDV), group A bovine rotavirus (BoRVA), and BCoV, targeting the 5′untranslated region [28], viral protein 6 gene [29], and nucleocapsid gene [9], respectively, according to the manufacturer’s instructions. The reaction mixture consisted of a total volume of 20 µL, containing 10 µL of 2× One Step TB Green RT-PCR Buffer, 0.4 µL of TaKaRa Ex Taq HS, 0.4 µL of PrimeScript RT Enzyme Mix, 0.4 µL of each primer, and 2 µL of template RNA. Reverse transcription was conducted at 42 °C for 5 min, followed by initial denaturation at 95 °C for 10 s. PCR amplification was performed for 40 cycles (denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 34 s). Each run included positive controls and a no-template control. Positive controls were as follows: BVDV and BoRVA were obtained from samples available in our laboratory [28, 29]. BCoV (BC94 strain) was provided by the Animal and Plant Quarantine Agency (AQA, Gimcheon, ROK) [30]. Amplification specificity was confirmed by melting curve analysis. In addition, amplified products were visualized on 1.5% agarose gels stained with ethidium bromide (Supplementary Fig. 1). Representative melting temperature (Tm) values were consistent for each virus, and the corresponding cycle threshold (Ct) and Tm values are presented in supplementary Table 1. Samples with a Ct value ≤ 30 were considered positive. Although amplification was performed for up to 40 cycles, Ct values > 30 were conservatively interpreted as low-level or non-specific signals to minimize false-positives. Conventional PCR was carried out for detection of Cryptosporidium parvum [31].

Of the pathogens tested, only BoRVA was detected in all intestinal tissues except the ileum, whereas BCoV was only present in the lungs (Supplementary Fig. 1). Additionally, lung tissues were tested for common respiratory viral pathogens. Bovine herpesvirus-1 [32] was tested by PCR, whereas bovine respiratory syncytial virus [33] and bovine parainfluenza virus type 3 [33] were examined by RT-PCR; all results were negative.

For histopathological examination, tissue samples were immediately fixed in 10% neutral-buffered formalin, transported to the Animal Disease Diagnostic Division of AQA (Gimcheon), and processed for hematoxylin−eosin (H&E) staining. Histopathological evaluation revealed mild bronchiolitis in the left lung and mild interstitial pneumonia in the right lung (Fig. 3).

Fig. 3.

Histopathological findings in the lungs. The calf showed mild bronchiolitis, including attenuation of the bronchiolar epithelial cells (hollow arrows) and infiltration by necrotic neutrophils and lymphocytes (arrow) in the left lung (A). This calf also showed mild interstitial pneumonia characterized by focal infiltrations of necrotic inflammatory cells within the alveolar interstitium and alveolar spaces (arrows) in the right lung (B). Arrows indicate the lesion sites. Scale bars: A, 20 μm; B, 50 μm

Immunohistochemistry (IHC) was also conducted to assess the presence of viral antigen in the lungs. Briefly, 5-µm paraffin sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. The sections were incubated with a mouse monoclonal anti-BCoV antibody (clone BC6-4, NDSU Veterinary Diagnostic Laboratory, Fargo, ND, USA; 1:500), followed by incubation with a universal HRP-conjugated secondary antibody (Ventana Medical Systems, Oro Valley, AZ, USA). Immunoreactive signals were visualized using a DAB substrate (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. Isotype-matched IgG served as a negative control. IHC analysis revealed BCoV antigen in bronchiolar epithelial cells of the left lung and in alveolar macrophages of the right lung (Fig. 4). An experienced veterinary pathologist evaluated the H&E slides and IHC staining.

Fig. 4.

IHC detection of BCoV antigens in the lungs. Positive immunostaining (brown) was observed in the bronchiolar epithelium of the left lung (A), the bronchiolar epithelium with mild bronchiolitis in the left lung (B), and alveolar macrophages of the right lung (C). Scale bars: A, 20 μm; B, 20 μm; C, 50 μm

Discussion and conclusions

This case report describes BCoV-associated pulmonary infection in a 7-day-old Holstein calf with diarrhea. BCoV antigen was detected exclusively in the lungs by real-time PCR and IHC, whereas all other tissues, including the gastrointestinal tract, tested negative. The calf had no respiratory clinical signs; however, BCoV antigen was detected within the pulmonary lesions, suggesting that the lesions might be associated with BCoV infection. At present, it remains unclear whether respiratory involvement was truly mild or whether diarrhea obscured subtle respiratory signs. Several studies have reported that respiratory BCoV is typically associated with mild respiratory disease or pneumonia in calves aged 2 weeks to 6 months. In these cases, BCoV is detected in nasal secretions and lungs and often also in the intestine and feces [4, 21, 34, 35]. In contrast, our findings indicate that BCoV-associated pulmonary infection can occur much earlier than previously recognized and may be confined to the respiratory tract, as evidenced by the absence of viral detection in the colon. The lack of respiratory signs in this calf might therefore reflect subclinical pulmonary infection.

Previous studies have demonstrated that BCoV has dual tropism for the respiratory and intestinal tracts, with experimental infections showing initial viral replication in the respiratory tract followed by dissemination to the intestine [8, 36, 37]. In this case, however, BCoV replication appeared restricted to the lungs, possibly because the calf was euthanized early before viral dissemination to the intestine or other organs. Several studies have reported that clinical manifestations of BCoV vary with age, with enteric disease predominating in neonatal calves, whereas respiratory signs are more common in older calves and feedlot cattle [6, 35]. Notably, an age-related analysis indicated that although respiratory clinical signs were most commonly observed in calves aged 61−180 days, BCoV was frequently detected in respiratory samples from younger calves, particularly those aged 0–7 days [35]. In addition, the detection rate of viral RNA was higher in the respiratory samples than in the enteric samples in young calves aged 8−30 days. Together, these findings suggest that BCoV respiratory infection can occur early in life without obvious respiratory signs and may involve the respiratory tract earlier than the intestinal tract. Such age-dependent differences in clinical presentation may relate to variation in immune maturity and passive immunity among calves. Based on our findings, BCoV infection may initially involve the respiratory tract before dissemination to the intestine or the onset of detectable fecal shedding. This is consistent with prior reports indicating that nasal shedding can precede fecal shedding during the course of BCoV infection [37]. Therefore, the absence of BCoV detection in the colon of this neonatal calf does not necessarily preclude active BCoV infection, particularly during the early phase of disease.

In this study, viral antigen was localized to bronchiolar epithelial cells and alveolar macrophages. The mild bronchiolitis and interstitial pneumonia observed by H&E staining were consistent with the distribution of viral antigen, indicating that the pulmonary lesions are closely associated with viral infection. These findings are consistent with previous reports demonstrating the presence of BCoV in respiratory epithelial cells and alveolar macrophages [17, 18, 38]. In experimental BCoV infections, inoculated calves failed to exhibit distinct respiratory clinical signs, but viral antigen was detectable in respiratory tissues, including the bronchial and tracheal epithelium and the alveolar interstitium, and was associated with histopathological lesions such as interstitial pneumonia and macrophage infiltration [18, 27, 38, 39]. By contrast, field studies of BRD have reported epithelial attenuation and bronchiolitis obliterans in BCoV-positive lungs, suggesting that BCoV may contribute to pulmonary lesion development [17]. Overall, the close correspondence between antigen localization and pulmonary lesions in this case supports a pathogenic role for BCoV in the development of respiratory diseases, even in the absence of clear respiratory clinical signs.

Multiple studies have confirmed that BCoV contributes to respiratory disease cases. In the United States, BCoV RNA has been detected in the bronchial and tracheal epithelium of feedlot cattle affected by BRDC [17]. In Italy, BCoV was identified in calves with severe respiratory distress and interstitial pneumonia during a BRDC outbreak [40]. Similarly, in Ireland, BCoV was the most frequently identified virus (22.9%) in nasal swabs from calves under 3 months of age [41]. In a large-scale epidemiological study of BRDC outbreaks in Spain, BCoV was the most frequently detected virus (39.7%), primarily affecting calves under 5 months of age [42]. Taken together, evidence from different geographic regions indicates that BCoV-associated BRD is not a uniform condition but spans diverse clinical and pathological patterns. In contrast, in the ROK, BCoV is primarily viewed as an enteric pathogen that causes diarrhea in cattle; its role as a respiratory pathogen has received less attention. The present case suggests that pulmonary BCoV infection can occur naturally in a neonatal calf in the ROK. This limited attention may partly reflect a lack of comprehensive investigations into BCoV as an etiological agent of BRDC in the ROK. Together, these findings provide additional insight into BCoV pathogenesis and underscore the need for further studies to define the prevalence and pathogenic potential of respiratory BCoV infection in cattle.

Globally, the prevalence of BCoV in BRDC cases ranges from 10% to more than 70%, across geographical regions [43]. Previous studies have linked BCoV seropositivity to higher risk of respiratory disease and poorer growth performance [44]. However, respiratory BCoV isolates have been reported to be antigenically indistinguishable from enteric BCoVs [20], suggesting that BCoV pathogenesis is multifactorial and involves host and environmental factors in addition to viral genetic characteristics [4]. In this case, BCoV infection was confined to the lungs of a neonatal calf, supporting BCoV’s capacity to cause pulmonary disease even without typical respiratory signs such as nasal discharge or cough. Further studies are needed to clarify the mechanisms underlying lung-restricted BCoV infection and to determine how host age, immune status, and environmental factors contribute to atypical disease presentations.

This report has several limitations. First, this is a single-case report, so the findings have very limited generalizability. Second, only selected agents were tested, and a comprehensive investigation of respiratory pathogens was not performed. Third, no bacteriological examination was performed; therefore, secondary or opportunistic bacteria associated with bovine respiratory disease complex cannot be excluded. Nevertheless, these findings provide supportive evidence for the potential involvement of BCoV in pulmonary lesions.

In conclusion, this case describes a naturally occurring pulmonary infection caused by BCoV, with viral antigen confined to the lungs despite no respiratory signs. These findings indicate that BCoV infection may occur in the respiratory tract at a very early age. Therefore, BCoV should be considered an important respiratory pathogen in newborn calves, and clinicians should include respiratory assessment in the diagnostic approach for young calves, even when diarrhea is the primary clinical sign.

Abbreviations

BCoV

Bovine coronavirus

BoRVA

Group A bovine rotavirus

BRDC

Bovine respiratory disease complex

BVDV

Bovine viral diarrhea virus

Ct

Cycle threshold

H&E

Hematoxylin−eosin

IHC

Immunohistochemistry

N

Nucleocapsid

ROK

Republic of Korea

RT-PCR

Reverse transcription-polymerase chain reaction

Tm

Melting temperature

Authors' contributions

KSC designed the study. HCC, JS and YK performed sampling and analyzed data. JK evaluated the H&E slides and IHC staining. HCC and KSC wrote the manuscript. All authors read and approved the final manuscript.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2025-25433218) (for HC Cho). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-23525211) (for KS Choi).

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

This study was exempt from ethical approval by the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University because the IACUC evaluates only laboratory animals housed in indoor facilities, not outdoor animals; therefore, ethics approval was not required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.