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Calf mortality constitutes a substantial loss for agriculture economy-based countries and is also a significant herd problem in developed countries. However, the occurrence and frequency of responsible gastro-intestinal (GI) pathogens in severe newborn diarrhea is still not well known. We aimed to determine the seasonal and age-associated pathogen distribution of severe diarrhea in newborn calves admitted to the intensive care unit (ICU) of Erciyes University animal hospital over a year. Fecal samples were collected during the ICU admissions, and specimens were subjected to a diarrheal pathogen screening panel that included bovine coronavirus (BCoV), Cryptosporidium spp., ETEC K99+, and bovine rotavirus, using RT-PCR and conventional PCR methods. Further isolation experiments were performed with permissive cell cultures and bacterial enrichment methods to identify the clinical importance of infectious pathogen shedding in the ICU. Among the hospitalized calves aged less than 45 days old, the majority of calves originated from small farms (85.9%). The pathogen that most frequently occurred was Cryptosporidium spp. (61.5%) followed by rotavirus (56.4%). The frequency of animal admission to ICU and GI pathogen identification was higher during the winter season (44.9%) when compared to other seasons. Most calves included in the study were 1–6 days old (44.9%). Lastly, co-infection with rotavirus and Cryptosporidium spp. occurred more frequently than other dual or multi-infection events. This study was the first to define severe diarrhea—causing GI pathogens from ICU admitted newborn calves in Turkey.

Background Calf diarrhea remains a significant concern in the global cattle industry, leading to considerable economic losses. Infectious pathogens are among the primary causes of this disease. In this study, the prevalence of 7 pathogens—bovine rotavirus (BRV), bovine coronavirus (BCV), bovine viral diarrhea virus (BVDV) types 1 and 2, Cryptosporidium parvum , Giardia spp., and Eimeria spp.—associated with calf diarrhea was investigated using polymerase chain reaction (PCR). A metagenomic approach was also applied to identify additional RNA viral pathogens from unknown causes of diarrheic fecal samples in the Republic of Korea (ROK). Results A total of 810 fecal samples from Hanwoo calves ( Bos taurus coreanae ) were collected, consisting of 526 normal samples (267 with a fecal score of 0 and 259 with a fecal score of 1) and 284 diarrheic samples (178 with a fecal score of 2 and 106 with a fecal score of 3). All 7 pathogens were detected by PCR in feces and their detection rates and mean fecal scores for each were as follows: BRV (14.0%, 1.41), BCV (3.2%, 1.42), BVDV1 (2.1%, 1.35), BVDV2 (4.9%, 1.33), C . parvum (9.8%, 1.66), Eimeria spp. (1.9%, 1.73), and Giardia spp. (0.9%, 0.71). Among these pathogens, BRV ( p  = 0.004), C . parvum ( p  < 0.001), and Eimeria spp. ( p  = 0.027) showed an increase in prevalence with higher fecal scores. Twenty-one fecal samples negative for all pathogens were randomly selected and subjected to high-throughput sequencing to identify RNA viral pathogens associated with calf diarrhea. This approach led to the identification of nearly complete genomic sequences for bovine astrovirus, bovine enterovirus, bovine kobuvirus, bovine nebovirus, bovine norovirus, bovine boosepivirus B, bovine parechovirus, bovine torovirus, C . parvum virus 1, and hunnivirus. Conclusions This study represents the first investigation of hunnivirus presence and provides a comprehensive description of the nearly complete genomes of 10 viruses associated with calf diarrhea in the ROK. The findings contribute to a better understanding of the epidemiology and molecular characteristics of calf diarrhea-associated pathogens in the ROK, highlighting the potential application of high-throughput sequencing for diagnosing other diseases.

Fecal microbiota transplantation (FMT) is a therapeutic modality for treating neonatal calf diarrhea. Several practical barriers, including donor selection, fecal collection, and a limited timeframe for FMT, are the main constraints to using fresh feces for implementing on-farm FMT. We report the utility of FMT with pretreated ready-to-use frozen (F) or freeze-dried (FD) microorganisms for treating calf diarrhea. In total, 19 FMT (F-FMT, n = 10 and FD-FMT, n = 9) treatments were conducted. Both FMT treatments were 100% clinically effective; however, multi-omics analysis showed that FD-FMT was superior to F-FMT. Machine learning analysis with SourceTracker confirmed that donor microbiota was retained four times better in the recipient calves treated with FD-FMT than F-FMT. A predictive model based on receiver operating characteristic curve analysis and area under the curve showed that FD-FMT was more discriminative than F-FMT of the observed changes in microbiota and metabolites during disease recovery. These results provide new insights into establishing methods for preparing fecal microorganisms to increase the quality of FMT in animals and may contribute to FMT in humans.

Viral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea. Specific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods. This novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region. The mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus; the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Calf diarrhea is a complex digestive disorder in cattle that imposes significant economic losses in terms of calf mortality, growth impairment, and treatment costs. Both infectious and non-infectious agents contribute to its aetiology; however, most of the infectious cases are caused by viruses, often accompanied by severe co-infections. To identify viral culprits, we performed viral metagenomic sequencing on three pooled samples from the 150 diarrheal samples from Xinjiang, China, which helped with identification of the following four predominant agents: bovine nepovirus (BNeV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and bovine enterovirus (BEV). Currently, the process of diagnosing these pathogens involves time-consuming workflows, limited sensitivity, poor portability, and lack of field applicability. Keeping these diagnostic shortcomings in mind, an integrated platform called RAA-CRISPR/Cas12a system was developed by combining recombinase-aided amplification (RAA) at 37°C with CRISPR/Cas12a-mediated fluorescence detection, which achieved 100–100,000 times higher sensitivity than conventional polymerase chain reaction (PCR) (detection limits: 1–10 copies/μL) and demonstrated 100% specificity against non-target pathogens. Clinical validation of sensitivity and specificity of 252 samples revealed 1.6–4.9 times higher detection rates (239 positives) than PCR (81 positives), which was consistent with PCR-confirmed cases. The assay’s 40-min. workflow enables rapid on-site deployment without specialized instrumentation, as it requires only a portable heat block and blue LED transilluminator. Hence, with its laboratory accuracy and field applicability, this method helps in early identification of pathogens, outbreak containment and mitigation of economic loss in the global cattle industry.

Background Viruses and the virome have received increased attention in the context of calf diarrhea and with the advancement of high-throughput sequencing the detection and discovery of viruses has been improved. Calf diarrhea, being the main contributor to calf morbidity and mortality, is a major issue within the livestock sector in Ethiopia. However, studies on viruses and the virome in calves is lacking in the country. Therefore, we utilized viral metagenomics to investigate the diversity of RNA viruses in healthy and diarrheic calves from central Ethiopia. Methods Fecal material from 47 calves were collected, pooled, and sequenced using Illumina. Following sequencing, the virome composition and individual viral sequences were investigated using bioinformatic analysis. Results The metagenomic analysis revealed the presence of several RNA viruses, including rotavirus and bovine coronavirus, known causative agents in calf diarrhea. In addition, several enteric RNA viruses that have not been detected in cattle in Ethiopia previously, such as norovirus, nebovirus, astrovirus, torovirus, kobuvirus, enterovirus, boosepivirus and hunnivirus were identified. Furthermore, a highly divergent viral sequence, which we gave the working name suluvirus, was found. Suluvirus showed a similar genome structure to viruses within the Picornaviridae family and phylogenetic analysis showed that it clusters with crohiviruses. However, due to its very divergent amino acid sequence, we propose that suluvirus represent either a new genus within the Picornaviridae or a new species within crohiviruses. Conclusions To our knowledge, this is the first characterization of the RNA virome in Ethiopian cattle and the study revealed multiple RNA viruses circulating in both diarrheic and healthy calves, as well as a putative novel virus, suluvirus. Our study highlights that viral metagenomics is a powerful tool in understanding the divergence of viruses and their possible association to calf diarrhea, enabling characterization of known viruses as well as discovery of novel viruses.

Bovine coronavirus (BCoV) is an important viral agent associated with neonatal calf diarrhea (NCD), winter dysentery, and bovine respiratory disease, contributing to economic losses in cattle production worldwide. To date, BCoV sequence data from Yanbian, Northeast China, are limited in public datasets. In this study, we report the first successful isolation of a BCoV strain (BCoV-YBYJ) from a diarrheic calf in Yanbian using MDBK cells and characterize its replication in vitro and describe exploratory colon and lung findings following high-dose oral exposure in a murine model. The isolate replicated efficiently in MDBK cells. In mice, viral RNA was detected in the colon and lung with mild-to-moderate histological changes under high-dose exposure (nonnatural host). Phylogenetic analysis based on partial ORF1a, S, and M sequences showed that BCoV-YBYJ clustered with contemporary Chinese isolates within the available reference set, supporting regional genetic relatedness based on partial loci. In parallel, three SYBR Green I-based RT-qPCR assays targeting BCoV, bovine rotavirus (BRV), and bovine parvovirus (BPV) were established and analytically validated. These assays demonstrated satisfactory analytical sensitivity, specificity, and reproducibility within the validated dynamic range, and BCoV RNA was detected in 70% (140/200) of targeted diarrheic calf specimens (  = 200). This study provides a Yanbian BCoV isolate and analytically validated RT-qPCR assays to support differential detection and surveillance of major viral agents associated with calf diarrhea. Because these were targeted submissions from diarrheic calves, this proportion is reported descriptively and should not be interpreted as population prevalence. The BRV and BPV assays underwent analytical validation only and require further clinical evaluation before routine diagnostic application.

Background Diarrhea is a major cause of morbidity and mortality in young calves, resulting in considerable economic loss for dairy farms. To determine if some gut microbes might have resistance to dysbiotic process with calf diarrhea by dictating the microbial co-occurrence patterns from birth to post-weaning, we examined the dynamic development of the gut microbiota and diarrhea status using two animal trials, with the first trial having 14 Holstein dairy calves whose fecal samples were collected 18 times over 78 d from birth to 15 d post-weaning and the second trial having 43 Holstein dairy calves whose fecal samples were collected daily from 8 to 18 days of age corresponding to the first diarrhea peak of trial 1. Results Metataxonomic analysis of the fecal microbiota showed that the development of gut microbiota had three age periods with birth and weaning as the separatrices. Two diarrhea peaks were observed during the transition of the three age periods. Fusobacteriaceae was identified as a diarrhea-associated taxon both in the early stage and during weaning, and Clostridium _ sensu_stricto_1 was another increased genus among diarrheic calves in the early stage. In the neonatal calves, Prevotella _ 2 (ASV4 and ASV26), Prevotella _ 9 (ASV43), and Alloprevotella (ASV14) were negatively associated with Clostridium_sensu_stricto_1 (ASV48), the keystone taxa of the diarrhea-phase module. During weaning, unclassified Muribaculaceae (ASV28 and ASV44), UBA1819 (ASV151), Barnesiella (ASV497) , and Ruminococcaceae _ UCG-005 (ASV254) were identified being associated with non-diarrheic status, and they aggregated in the non-diarrhea module of co-occurrence patterns wherein unclassified Muribaculaceae (ASV28) and Barnesiella (ASV497) had a direct negative relationship with the members of the diarrhea module. Conclusions Taken together, our results suggest that the dynamic successions of calf gut microbiota and the interactions among some bacteria could influence calf diarrhea, and some species of Prevotella might be the core microbiota in both neonatal and weaning calves, while species of Muribaculaceae might be the core microbiota in weaning calves for preventing calf diarrhea. Some ASVs affiliated with Prevotella_2 (ASV4 and ASV26), Prevotella_9 (ASV43), Alloprevotella (AVS14), unclassified Muribaculaceae (ASV28 and ASV44), UBA1819 (ASV151), Ruminococcaceae _ UCG-005 (ASV254), and Barnesiella (ASV497) might be proper probiotics for preventing calf diarrhea whereas Clostridium_sensu_stricto_1 (ASV48) might be the biomarker for diarrhea risk in specific commercial farms.

Neonatal calf diarrhea (NCD) is a major cause of morbidity and mortality in calves, prompting the development of effective vaccines. This study aimed to formulate an inactivated combined vaccine candidate against Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV) using a single Aluminum Hydroxide adjuvant combined with Saponin to evaluate immune responses in mice. BRV and BCoV antigens were inactivated following multiplication in MA-104 and MDBK cell lines, respectively, and inactivated with 0.1 M BEI. The inactivation process was confirmed through CPE observations and real-time qPCR results from three blind passages. Particle sizes and densities of BRV and BCoV antigens were assessed using nanoparticle analysis, confirming that both viruses exhibited optimal particle characteristics. The immunogenicity of the resulting vaccines was evaluated through serological and cellular immunity assays. In ELISA and serum neutralization tests, all vaccine groups containing BRV and BCoV displayed significantly higher antibody responses compared to the control group (p < 0.05). Notably, the combined BRV and BCoV vaccine groups exhibited a strong neutralization effect. Cytokine responses, including IFN-ɣ, IL-2, IL-4, IL-6, and TNF-α, were significantly increased in the vaccine groups compared to the control group (p < 0.05). These findings suggest that combined vaccines containing BRV and BCoV antigens hold the potential to induce an effective immune response. Previous studies have highlighted the beneficial effects of Quil-A® on cellular immunity, and our results further support its role in enhancing both humoral and cellular immune responses across a broad range of individuals. •An inactivated combined BRV and BCoV vaccine was formulated with ALOOH and Quil-A®.•BEI-inactivated antigens were validated by TCID₅₀, CPE, and real-time qPCR.•Nanoparticle analysis confirmed optimal particle sizes for BRV and BCoV.•Combined vaccine induced strong humoral and cellular responses in mice.•Results guide future vaccine safety and efficacy studies in neonatal calves.