BACKGROUND: Cupriavidus gilardii is an aerobic, gram-negative, motile, glucose-nonfermenting bacillus, first described in 1999. Typically, it exhibits low pathogenicity in humans, causing opportunistic infections primarily in individuals with compromised immune systems. This bacterium has been also found in various environmental sources such as plants and contaminated soils. Notably, there have been no documented cases of C. gilardii infections in animals.
METHODS: This case report outlines a bovine neonatal diarrhea outbreak that occurred in Northern Greece, during which C. gilardii was isolated. Faecal samples from 5-day-old calves were collected and transported to the laboratory for further examination. Bacterial culture and next generation sequencing techniques were employed to confirm the presence of this bacterium in the samples. Following the isolation and identification of C. gilardii from the samples, an autogenous vaccine was produced and administered to the cows within the farm. Subsequent to vaccination, a progressive reduction in calf diarrhea and deaths was observed, leading to their eventual complete resolution. To the best of our knowledge, this represents the first documentation of C. gilardii isolation from cases of bovine neonatal diarrhea.
CONCLUSIONS: This case report presents the first isolation case of C. gilardii from animal samples and more specifically from calf faecal samples. It represents an important observation, providing evidence that this opportunistic human pathogen could contribute to clinical symptoms in animals.

The objective of this study was to explore the dynamic changes in the gut microbiota of Simmental calves before weaning and to compare the microbial composition and functionality between healthy calves and those with diarrhea. Fourteen neonatal Simmental calves were divided into a healthy group (n = 8) and a diarrhea group (n = 6). Rectal stool samples were collected from each calf on days 1, 3, 5, 7, 9, 12, 15, 18, 22, 26, 30, 35, and 40. High-throughput sequencing of the 16S rRNA gene V1-V9 region was conducted to examine changes in the gut microbiota over time in both groups and to assess the influence of diarrhea on microbiota structure and function. Escherichia coli, Bacteroides fragilis, and B. vulgatus were the top three bacterial species in preweaning Simmental calves. Meanwhile, the major functions of the fecal microbiota included \"metabolic pathways\", \"biosynthesis of secondary metabolites\", \"biosynthesis of antibiotics\", \"microbial metabolism in diverse environments\", and \"biosynthesis of amino acids\". For calves in the healthy group, PCoA revealed that the bacterial profiles on days 1, 3, 5, 7, and 9 differed from those on days 15, 18, 22, 26, 30, 35, and 40. The profiles on day 12 clustered with both groups, indicating that microbial structure changes increased with age. When comparing the relative abundance of bacteria between healthy and diarrheic calves, the beneficial Lactobacillus johnsonii, Faecalibacterium prausnitzii, and Limosilactobacillus were significantly more abundant in the healthy group than those in the diarrhea group (p < 0.05). This study provides fundamental insights into the gut microbiota composition of Simmental calves before weaning, potentially facilitating early interventions for calf diarrhea and probiotic development.

Little is known about shifts in the fecal microbiome of dairy calves preceding and following the incidence of gastrointestinal disease. The objective of this cohort study was to describe the fecal microbiome of preweaned dairy calves before, during, and after gastrointestinal disease. A total of 111 Holstein dairy calves were enrolled on 2 dairies (D1 and D2) and followed until 5 weeks old. Health assessments were performed weekly and fecal samples were collected every other week. Of the 111 calves, 12 calves from D1 and 12 calves from D2 were retrospectively defined as healthy, and 7 calves from D1 and 11 calves from D2 were defined as diarrheic. Samples from these calves were sequenced targeting the 16S rRNA gene and compared based on health status within age groups and farms: healthy (0-1 week old) vs. pre-diarrheic (0-1 week old), healthy (2-3 weeks old) vs. diarrheic (2-3 weeks old), and healthy (4-5 weeks old) vs. post-diarrheic (4-5 weeks old) calves. Healthy and diarrheic samples clustered together based on age rather than health status on both farms. Based on linear discriminant analysis, a few species were identified to be differently enriched when comparing health status within age groups and farm. Among them, Bifidobacterium sp. was differently enriched in pre-diarrheic calves at D1 (0-1 week old) whereas healthy calves of the same age group and farm showed a higher abundance of Escherichia coli. Bifidobacterium sp. was identified as a biomarker of fecal samples from healthy calves (2-3 weeks old) on D1 when compared with diarrheic calves of the same age group and farm. Feces from diarrheic calves on D2 (2-3 weeks old) were characterized by taxa from Peptostreptococcus and Anaerovibrio genera whereas fecal samples of age-matched healthy calves were characterized by Collinsella aerofaciens and Bifidobacterium longum. After resolution of uncomplicated diarrhea (4-5 weeks old), Collinsella aerofaciens was more abundant in D2 calves whereas Bacteriodes uniformis was more abundant in D1 calves. Taken together, these findings suggest that the age of the preweaned calf is the major driver of changes to fecal microbiome composition and diversity even in the face of uncomplicated gastrointestinal disease.

This work aims to: (1) elucidate the immune response exhibited by CD4 + and CD8 + T lymphocyte cells in response to various infectious agents in calves suffering with neonatal diarrhea; and (2) determine and investigate the association between serum selenium levels and T lymphocyte subtypes in neonatal calves afflicted with neonatal diarrhea and infected with various infectious agents. The study encompassed a cohort of 50 calves, encompassing both sexes and various breeds, within the neonatal age range (1-28 days old). Subdivided into distinct groups, the calves were categorized based on the causative agents of neonatal diarrhea, including Rotavirus (n = 10), Cryptosporidium parvum (C.parvum) (n = 10), Coronavirus (n = 5), Rotavirus+C.parvum (n = 5), and a Control group (n = 20). Blood samples were meticulously obtained from the vena jugularis of all animals utilizing specific techniques-8 ml in tubes devoid of anticoagulant and 3 ml in blood collection tubes containing EDTA. Serum selenium levels were analyzed by ICP-MS. Flow Cytometry device was used to determine CD4 + and CD8 +T lymphocyte levels. In this study, although there was no statistically significant difference in serum selenium levels between all study groups, it was found that the selenium level in the control group was not sufficient. CD4 T lymphocyte levels, the rotavirus+C.parvum group exhibited a statistically significant elevation compared to the coronavirus group. Regarding CD8 + T lymphocyte levels, the coronavirus group demonstrated a statistically significant increase when compared to the control group. In intragroup analyses of CD8 + T lymphocyte levels, the coronavirus group exhibited a significant elevation compared to the rotavirus group, C.parvum group, and the C.parvum + Rotavirus group. A significant negative correlation was detected between selenium levels and CD4 + T lymphocytes, while no correlation was found between CD8 + T lymphocytes. Fibrinogen concentration exhibited statistical significance, being higher in the Rotavirus group (p < 0.008) compared to the control group, in the C.parvum group (p < 0.004) compared to the control group, and in the Coronavirus group (p < 0.001) compared to the control group. The leukocyte count demonstrated statistical significance, being higher in the Rotavirus group compared to the control group (p < 0.001), in the Rotavirus+C.parvum group compared to the control group (p < 0.002), and in the Coronavirus group compared to the control group (p < 0.011). In conclusion, the data derived from this study illuminate discernible disparities in CD4 + and CD8 + T lymphocyte immune responses, contingent upon the specific etiological agent associated with neonatal diarrhea. Furthermore, the study underscores the importance of considering selenium deficiency as a relevant factor in calves affected by neonatal diarrhea.

UNASSIGNED: Bovine coronaviruses (BCoVs) are zoonotic diseases that result in substantial economic losses due to mortality, impaired growth, and increased medication expenses in large animals. These viruses pose a risk to children who live beside infected animal, as they can cause diarrhea. This study was dedicated to molecular and antigen detections and phylogenetic and immunoinformatics analysis of zoonotic coronavirus (CoV) in Iran.
UNASSIGNED: A total of 77 diarrheic samples were collected from Holstein dairy herds in selected provinces of Iran. Samples were tested by capture antibody enzyme-linked immunosorbent assay (ELISA) to detect CoV and reverse transcriptase-polymerase chain reaction (RT-PCR) for verification of detection and also genotyping of spike glycoprotein in CoV-positive samples. After statistical analysis, nucleotide sequence alignment, and nucleotide and protein phylogenetic tree construction, the centralized sequence for vaccine strains was obtained using computationally optimized broadly reactive antigen (COBRA)\'s center-of-the-tree (COT) method.
UNASSIGNED: Twenty-two (28.5%) and eight (10.3%) of 77 samples were positive according to RT-PCR and ELISA, respectively. (Basic Local Alignment Search Tool) BLAST and phylogenetic analysis revealed that most similar sequences to the Iranian CoV sequence were for European countries. Furthermore, there were strong correlations to other CoVs in humans and wild and domesticated animals. As CoV has variable COT, the most recent strains and COBRA vaccine strains were obtained.
UNASSIGNED: Based on the high prevalence of this viral disease in calves and its economic impact on the breeding industry, as well as the potential transmission to humans and correlation with World Health Organization (WHO) One Health approach guidelines, the study emphasizes the importance of implementing preventive strategies such as animal vaccination.

The objectives of this study were to evaluate factors associated with colostrum quality and FTPI in calves from dairy farms in Austria and to assess the associations between disease occurrence and FTPI in calves. In total, 250 calves and their colostrum samples originating from 11 dairy farms were included in the study. All calves born between September 2021 and September 2022 were included. Blood samples were collected between the third and the sixth day of age. The farmers were trained in disease detection and recorded any health events within the first three weeks of age daily. Multiparous cows (>3 lactation) and colostrum harvesting within the first 2 hours after parturition were significantly associated with good colostrum quality (>22% Brix). Colostrum quantity (≥2 L) and quality (≥22% Brix) acted as protective factors against FTPI (serum Brix ≥ 8.4%) with odds ratios of OR = 0.41 and OR = 0.26, respectively. Calves facing any health event (diarrhea, navel illness, bovine respiratory disease, abnormal behavior) in the first three weeks of life had a higher probability of FTPI. Calves exhibiting diarrhea in the first 3 weeks of life were associated with having FTPI (OR = 2.69). The results confirm the current recommendations for good colostrum management practices and the impact of FTPI on calf morbidity.

Oxidative stress is the imbalanced redox status between oxidant production and their scavengers leading to intestinal physiological dysfunction. However, the role of systemic and local oxidative status during neonatal calf diarrhea is not known. This study assessed systemic (serum) and local (fecal) oxidative status when calves either naturally developed diarrhea or naturally recovered. Healthy calves were enrolled in the study at d 18 of age, and their health status was monitored from the enrollment. Based on their enteric health status on d 21 and 28, calves were grouped as continuous diarrhea from d 21 to 28 (n = 14), diarrhea at d 21 but recovered at d 28 (DH group, n = 19), healthy at d 21 but developed diarrhea at d 28 (HD group, n = 15), and healthy throughout the study (HH group, n = 16). Serum and fecal samples were collected at d 21 and 28 from all calves in the morning 2 h after feeding. Dynamics of oxidative stress indicators including reactive oxygen species (ROS), malondialdehyde (MDA), H2O2, 8-hydroxy-2\'-deoxyguanosine (8-OHDG), glutathione peroxidase, superoxide dismutase, catalase (CAT), and total antioxidant capacity and inflammatory indicators TNF-α, IL-1β, IL-4, IL-6, IL-10, and IFN-γ were evaluated using serum samples. In addition, fecal oxidative stress indicators ROS and MDA were measured. Serum ROS, MDA, 8-OHDG, as well as fecal ROS and MDA, were higher, whereas serum CAT and H2O2 were lower in diarrheic calves than those of healthy calves. Serum ROS, MDA, and 8OHDG and fecal ROS and MDA increased in the HD group from d 21 to 28 as they developed diarrhea. In contrast, all these oxidative stress markers decreased in the DH group from d 21 to 28 as they recovered. However, serum H2O2 had an opposite changing trend, which became lower in the HD group and higher in the DH group at d 28. In conclusion, both systemic and local oxidative stress markers and cytokine profiles altered as calves moved from being healthy to having diarrhea or vice versa. Serum ROS, MDA, and 8-OHDG can be used to develop biomarkers to screen calves prone to enteric infections during the preweaning period.

Quantifying the water and mineral losses in feces is essential to determine the optimal composition of oral rehydration solutions (ORS) for diarrheic animals. In a randomized complete block design, this study evaluated water, mineral, and blood acid-base balance of calves with naturally occurring diarrhea receiving ORS or a placebo. On d 0, 45 calves (age: 18 ± 3.2 d; mean ± SD) were selected based on the presence of visual signs of diarrhea, such as dirty tail or wet feces, along with clinical symptoms evaluated by measuring the skin turgor and the degree of enophthalmos. On d 1, calves were divided into blocks of 3 animals based on blood base excess (BE) measured at 0900 h, and within each block, calves were randomly assigned to 1 of 3 treatments (15 calves per treatment) including (1) a hypertonic ORS (HYPER; Na+ = 110 mmol/L; 370 mOsm/kg; strong ion difference [SID] = 60 mEq/L), (2) a hypotonic ORS with low Na+ (HYPO; Na+ = 77 mmol/L; 278 mOsm/kg; SID = 71 mEq/L), and (3) a placebo consisting of lukewarm water with 5 g/L of whey powder (CON). Milk replacer (MR) was fed through teat buckets twice daily at 0630 h and 1700 h in 2 equally sized meals of 2.5 L from d 1 to 3 and of 3.0 L on d 4 and 5. Treatments consisting of 2.0 L lukewarm solutions were administered between milk meals from d 1 to 3 at 1200 h and 2030 h through teat buckets. Refusals of MR and treatments were recorded daily, and blood samples were collected from the jugular vein once daily at arrival in the afternoon of d 0 and at 0900 h from d 1 to 5 after arrival. Urine and feces were collected quantitatively over a 48-h period from 1200 h on d 1 to 1200 h on d 3, and a representative sample of each 24-h period was stored. In addition, the volume of extracellular fluid was evaluated on d 2 by postprandial sampling over a 4-h period relative to the injection of sodium thiosulfate at 1300 h. Total daily fluid intake (MR, treatment, and water) from d 1 to 3 was greater in HYPER (LSM ± SEM; 8.9 ± 0.36 L/d) and HYPO (7.8 ± 0.34 L/d) than in CON (6.6 ± 0.34 L/d). This resulted in a greater water balance (water intake - fluid output in urine and feces) in calves receiving ORS (59.6 ± 6.28 g/kg BW per 24 h vs. 39.6 ± 6.08 g/kg BW per 24 h). Fecal Na+ losses were greater in HYPER than in the other treatments (81 ± 12.0 mg/kg BW per 24 h vs. 24 ± 11.8 mg/kg BW per 24 h). Blood pH was higher in HYPO (7.41 ± 0.016) than CON (7.35 ± 0.016) over the 5 monitoring days, whereas HYPER (7.37 ± 0.017) did not differ with other treatments. In this experimental model, diarrheic calves were likely unable to absorb the high Na+ load from HYPER, resulting in greater Na+ losses in feces, which might have impaired the alkalinizing capacity of HYPER. In contrast, HYPO significantly sustained blood acid-base balance compared with CON, whereas HYPER did not. This suggests that low tonicity ORS with a high SID are more suitable for diarrheic calves.

OBJECTIVE: Calf diarrhea is of great concern to the global dairy industry as it results in significant economic losses due to lower conception rates, reduced milk production, and early culling. Although there is evidence of an association between altered gut microbiota and diarrhea, remarkably little is known about the microbial and metabolic mechanisms underlying the link between gut microbiota dysbiosis and the occurrence of calf diarrhea. Here, we used fecal metagenomic and metabolomic analyses to demonstrate that gut microbiota-driven metabolic disorders of purine or arachidonic acid were associated with calf diarrhea. These altered gut microbiotas play vital roles in diarrhea pathogenesis and indicate that gut microbiota-targeted therapies could be useful for both prevention and treatment of diarrhea.

The changes in the composition of intestinal microbiota and metabolites have been linked to digestive disorders in calves, especially neonatal calf diarrhea. Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are known to be the primary culprits behind neonatal calf diarrhea. In this study, we analyzed changes in the fecal microbiota and metabolites of calves with neonatal diarrhea associated with BRV and BCoV infection using high-throughput 16S rRNA sequencing and metabolomics technology. The microbial diversity in the feces of calves infected with BRV and BCoV with diarrhea decreased significantly, and the composition changed significantly. The significant increase of Fusobacterium and the reductions of some bacteria genera, including Faecalibacterium, Bifidobacterium, Ruminococcus, Subdoligranulum, Parabacteroides, Collinsella, and Olsenella, etc., were closely related to diarrhea associated with BRV and BCoV infection. Metabolites in the feces of BRV and BCoV-infected calves with diarrhea were significantly changed. Phosphatidylcholine [PC; 16:1(9 Z)/16:1(9 Z)], lysophosphatidylethanolamine (LysoPE; 0:0/22:0), lysophosphatidylcholine (LysoPC; P-16:0) and LysoPE (0:0/18:0) were significantly higher in the feces of BRV-infected calves with diarrhea. In contrast, some others, such as desthiobiotin, were significantly lower. BRV infection affects glycerophospholipid metabolism and biotin metabolism in calves. Two differential metabolites were significantly increased, and 67 differential metabolites were significantly reduced in the feces of BCoV-infected calves with diarrhea. Seven significantly reduced metabolites, including deoxythymidylic acid (DTMP), dihydrobiopterin, dihydroneopterin triphosphate, cortexolone, cortisol, pantetheine, and pregnenolone sulfate, were enriched in the folate biosynthesis, pantothenate and CoA biosynthesis, pyrimidine metabolism, and steroid hormone biosynthesis pathway. The decrease in these metabolites was closely associated with increased harmful bacteria and reduced commensal bacteria. The content of short-chain fatty acids (SCFAs) such as acetic acid and propionic acid in the feces of BRV and BCoV-infected calves with diarrhea was lower than that of healthy calves, which was associated with the depletion of SCFAs-producing bacteria such as Parabacteroides, Fournierella, and Collinsella. The present study showed that BRV and BCoV infections changed the composition of the calf fecal microbiota and were associated with changes in fecal metabolites. This study lays the foundation for further revealing the roles of intestinal microbiota in neonatal calf diarrhea associated with BRV and BCoV infection.