Detection of the dominant pathogens in diarrheal calves of Ningxia, China in 2021-2022.

Introduction: Calf diarrhea is a complex disease that has long been an unsolved problem in the cattle industry. Ningxia is at the forefront of China in the scale of cattle breeding, and calf diarrhea gravely restricts the development of Ningxia's cattle industry. Methods: From July 2021 to May 2022, we collected diarrhea stool samples from calves aged 1-103 days from 23 farms in five cities in Ningxia, and performed PCR using specific primers for 15 major reported pathogens of calf diarrhea, including bacteria, viruses, and parasites. The effect of different seasons on the occurrence of diarrhea in calves was explored, the respective epidemic pathogens in different seasons were screened, and more detailed epidemiological investigations were carried out in Yinchuan and Wuzhong. In addition, we analyzed the relationship between different ages, river distributions and pathogen prevalence. Results: Eventually, 10 pathogens were detected, of which 9 pathogens were pathogenic and 1 pathogen was non-pathogenic. The pathogens with the highest detection rate were Cryptosporidium (50.46%), Bovine rotavirus (BRV) (23.18%), Escherichia coli (E. coli) K99 (20.00%), and Bovine coronavirus (BCoV) (11.82%). The remaining pathogens such as Coccidia (6.90%), Bovine Astrovirus (BoAstV) (5.46%), Bovine Torovirus (BToV) (4.09%), and Bovine Kobuvirus (BKoV) (3.18%) primarily existed in the form of mixed infection. Discussion: The analysis showed that different cities in Ningxia have different pathogens responsible for diarrhea, with Cryptosporidium and BRV being the most important pathogens responsible for diarrhea in calves in all cities. Control measures against those pathogens should be enforced to effectively prevent diarrhea in calves in China.

An end-point multiplex PCR/reverse transcription-PCR for detection of five agents of bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is frequently associated with single or mixed viral, bacterial and/or protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each potential agent; a time-consuming, laborious and expensive process. Herein, we describe an end-point multiplex PCR/reverse transcription-PCR (RT-PCR) for detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we optimized multiplex PCR/RT-PCR conditions. Next, we evaluated the analytical sensitivity of the assay and assessed the performance of the reaction by testing 95 samples of diarrheic calf feces. The analytical specificity was evaluated against bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our assay was about 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, about 5 × 10-4 CFU for S. enterica, 5 × 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No non-specific amplification of other bovine diarrhea agents was detected. Out of 95 samples analyzed, 50 were positive for at least one target, being 35 single and 15 mixed infections. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was the most frequent in mixed infections (11/15). Positive and negative multiplex results were confirmed in individual reactions. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster and easier NCD diagnosis, which may be useful for routine diagnosis and surveillance studies.

The Quinolone Resistance Genes in the Bacteriophage DNA Fractions in the Healthy Calf Stool Samples Via qPCR

The One Health approach shows that people, animals, plants, and environmental factors can affect each other. Phages are one of the mobile genetic elements. Quinolones are a critical group of antibiotics for both human and animal health and monitoring their antimicrobial resistance is very important. The aim of the study is to determine the frequency of the quinolone resistance gene in bacteriophage DNA fractions obtained from healthy calf stool samples. In our study, 50 samples from 6-9 months old calves, which were found to be healthy and not treated with any group of antibiotics in Sanliurfa province, were included. DNA isolation was made from phage lysates of stool samples and specific primers were used qnrA, qnrB and qnrS genes. qPCR was performed on LightCycler480. Despite not receiving any antibiotic treatment, qnrB was the most detected gene among the phage DNA fractions detected in 11 calves. While qnrA, qnrB and qnrS quinolone resistance genes were detected together in one sample, qnrB and qnrS resistance genes were found together in two samples. Our data, obtained from the study in Turkiye to search for antimicrobial resistance genes in phage fractions, showed the importance of the One Health approach and determined that it was highly effective in quinolone resistance gene shedding in healthy calves that had never been treated with antibiotics. It has been concluded that in empirical treatment with quinolone, attention should be paid to all living things and unconscious antibiotic use may cause the spread of resistance genes more than expected.Copyright © 2023, Veteriner Fakultesi Dergisi. All rights reserved.

Multiplex RT-qPCR Application in Early Detection of Bovine Respiratory Disease in Healthy Calves.

Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.

The relationship between serum cytokine profile and vitamin D in calves with neonatal diarrhea.

It is important to know the characteristics of the immunological response in newborn calf diarrhea, which is often caused by bacterial, viral and protozoal pathogens. Cytokinesare proteins that serve as chemical messengers to regulate theinnate and adaptive arms of theimmune response. Changes in circulatory cytokine levels provide valuable information for understanding the pathophysiological process and monitoring disease progression and inflammation. Vitamin D has important immunomodulatory effects, which include enhancing the innate immune system and inhibiting adaptative immune responses. The objective of this study was to evaluate the relationship between serum cytokine profile and vitamin D level in neonatal calves with diarrhea. The study population was comprised of 40 neonatal calves, 32 of which had diarrhea and 8 of which were healthy calves. The calves with diarrhea were allocated to four groups according to bacterial (Escherichia coli), viral (Rotavirus, Coronavirus) and protozoal (Cryptosporidium parvum) etiologies. Circulatory vitamin D metabolites (25-hydroxyvitamin D, 1,25-dihydroxyvitamin D) and cytokines (TNF-α, IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and IL-17) in the calves were determined. There was no statistically significant difference among the groups in 25-hydroxyvitamin D levels. 1,25-dihydroxyvitamin D levels were higher in Coronavirus and E. coli groups compared to the controls. Serum levels of all cytokines except for IL-13, were higher in E. coli group than those of the control group. As a result, differences in serum cytokines and vitamin D levels according to etiological factors in calf diarrhea indicate that vitamin D may play a role in the immune response in the disease.

Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves.

Bovine Coronavirus (BCoV) is a major pathogen associated with neonatal calf diarrhea. Standard practice dictates that to prevent BCoV diarrhea, dams should be immunized in the last stage of pregnancy to increase BCoV-specific antibody (Ab) titers in serum and colostrum. For the prevention to be effective, calves need to suck maternal colostrum within the first six to twelve hours of life before gut closure to ensure a good level of passive immunity. The high rate of maternal Ab transfer failure resulting from this process posed the need to develop alternative local passive immunity strategies to strengthen the prevention and treatment of BCoV diarrhea. Immunoglobulin Y technology represents a promising tool to address this gap. In this study, 200 laying hens were immunized with BCoV to obtain spray-dried egg powder enriched in specific IgY Abs to BCoV on a large production scale. To ensure batch-to-batch product consistency, a potency assay was statistically validated. With a sample size of 241, the BCoV-specific IgY ELISA showed a sensitivity and specificity of 97.7% and 98.2%, respectively. ELISA IgY Abs to BCoV correlated with virus-neutralizing Ab titers (Pearson correlation, R2 = 0.92, p < 0.001). Most importantly, a pilot efficacy study in newborn calves showed a significant delay and shorter duration of BCoV-associated diarrhea and shedding in IgY-treated colostrum-deprived calves. Calves were treated with milk supplemented with egg powder (final IgY Ab titer to BCoV ELISA = 512; VN = 32) for 14 days as a passive treatment before a challenge with BCoV and were compared to calves fed milk with no supplementation. This is the first study with proof of efficacy of a product based on egg powder manufactured at a scale that successfully prevents BCoV-associated neonatal calf diarrhea.

Molecular Characterization of Bovine Rotaviruses and Coronaviruses in Diarrheic Calves in Egypt (2014-2019)

Bovine Rotaviruses group A (BRVA) and Bovine Coronaviruses (BCoV) are the most prevalent viral agent worldwide in diarrheic calves aged less than 6 weeks, causing economic losses due to retarded growth, increased susceptibility to other infections, treatment cost, and calf mortalities. This study aimed to detect and molecularly characterize BRVA and BCoV from diarrhetic calves. A total of 82 fecal samples were collected from calves aged less than one month from three Egyptian governorates (Alexandria, Ismailia, and Sharqia). All fecal samples were tested for BRVA and BCoV by using probe based quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Consequently, positive samples contain relatively high viral genomic load were examined by RT-PCR for amplification of viral protein 7 (VP7) and viral protein 4 (VP4) genes (G- and P- typing) for BRV and full length S1 gene for BCoV. Out of 82 of tested samples, 14 (17.1%) and 22 (26.8%) were positive by qRT-PCR for BRV and BCoV, respectively. Only three and five samples had relatively high genomic load for BRV and BCoV, respectively for further testing by RT-PCR. BRV G-type was found in two samples and P-type was detected in one sample. The sequence analysis and phylogenetic tree typed these positive samples as P11 and G10. The sequences and phylogenetic analysis of BCoV positive strains (n=5) showed closely related viruses to each other and similar to previously characterized strains in Egypt since 2014. Further studies are required to antigenically characterize the circulating BRV and BCoV in Egypt.

Genetic Diversity of Bovine Group A Rotavirus Strains Circulating in Korean Calves during 2014 and 2018.

The purpose of this study was to investigate annual changes in BoRVA strains by examining the VP4 and VP7 genes of rotaviruses in Korean calves. Between 2014 and 2018, 35 out of 138 samples of calf diarrhea feces collected nationwide were positive for BoRVA. Further genetic characterization of the VP7 and VP4 genes of 35 BoRVA isolates identified three different G-genotypes (G6, G8, and G10) and two different P genotypes (P[5] and P[11]). The G6 genotype was most common (94.3%) in BoRVA-positive calves, followed by the P[5] genotype (82.9%). Four genotypes comprised combinations of VP4 and VP7: 80% were G6P[5], 14.2% were G6P[11], 2.9% were G8P[5], and 2.9% were G10P[11]. Susceptibility to infection was highest in calves aged < 10 days (35%) and lowest in calves aged 30−50 days (15.4%). The data presented herein suggest that the G6P[5] genotype is the main causative agent of diarrhea in Korean calves. In addition, it is predicted that G6P[5] will continue to act as a major cause of diarrhea in Korean calves.

Physical Activity of Obese Children and Adolescents with the Nonalcoholic Fatty Liver Disease during the Covid-19 Pandemic

Introduction: Social isolation due to the COVID-19 pandemic leads to changes in general physical activity in children with nonalcoholic fatty liver disease (NAFLD) which may aggravate the course of the disease and alleviate the efficacy of the treatment. Aims & Methods: The aim of our study was to investigate the physical activity (PA) in children and adolescents with NAFLD during the COVID-19 pandemic and its association with liver fibrosis. 40 obese patients with NAFLD aged from 10 to 17 years (average age was 12.15 +/- 2.51 years) were examined from September-October 2021. Obesity was established by body mass index (BMI) calculation and comparison with the sigma deviations of BMI values according to age and sex. The presence of liver fibrosis and steatosis was evaluated by transient elastography (Fibroscan502touch, France). Children were divided into 4 groups according to transient elastography and BMI: 1 group - 13 children with NAFLD and liver fibrosis, 2 group - 13 children with NAFLD without fibrosis, 3 group - 14 obese children without NAFLD and fibrosis. The 4 group (control) consisted of 10 children with normal weight without NAFLD and fibrosis. The assessment of physical activity was conducted with the Physical Activity Questionnaire for older children (PAQ-C) and adolescents (PAQ-A). Result(s): The final summary score of the PA amounted to 2.4+/-0.3 in the 1 group, 2.2+/-0.2 in the 2 group, 2.2+/-0.3 in the 3 group, 2.4+/-0.2 in the 4 group without significant differences between the groups. The level of PA in spare time was the lowest in all groups compared to other types of activity. The highest rate of the PA score was observed in all groups during physical education classes, but the number of children who attended these classes not regularly was 43.9% among whom do not attend physical education classes at all 9.8%, almost never - 2.4%, from time to time - 31.7% of children. Only 26.8% of patients were active at recess while 73.2% of children stood or walked within the classroom or sat down. The level of PA of chil dren right after school increased slightly, also without significant differences between groups, but children with liver fibrosis had the lowest PA level (2.1 +/-0.2). 24.4% of children did not have any PA right after school. Free time at the weekend was not accompanied by an increase in physical activity, on the contrary, the summary score decreased in almost all groups to 1.94 points, and the portion of children without physical activity remains stable (24.4%). According to self-reports children of the 1-3 groups had a lower level of physical activity score compared to children of the control group. Almost 73.2% of interviewed children understood that their level of physical activity was low. The total level of physical activity on each day of the week was the lowest in children with liver fibrosis (1 group). The highest percentage of PA absence was on weekends. The total PA score was negatively correlated with calf circumference (r = -0.582, p = 0.018), self-report PA tended to a negative correlation with the level of alaninaminotransferase (r = - 0.372, p = 0.056). Conclusion(s): Physical activity of obese NAFLD children during the COVID- 19 pandemic is low, especially in spare time, and does not rise at the weekends. The majority of children (73.2%) are inactive at recess as well as self-reported low level of PA. NAFLD children with liver fibrosis have the lowest total level of PA right after school and generally on each day of the week, which may reflect an insufficiency of adaptation.

Isolation and Genetic Characterization of a Bovine Coronavirus KBR-1 Strain from Calf Feces in South Korea.

Bovine coronavirus (BCoV) causes severe diarrhea in neonatal calves, winter dysentery in adult cattle, and respiratory disease in feedlot cattle, resulting in economic losses. A total of 16/140 calf diarrheic feces samples collected in South Korea between 2017 and 2018 were positive for BCoV. Phylogenetic analysis of the complete spike and hemagglutinin/esterase genes revealed that the 16 Korean BCoV strains belonged to group GIIa along with Korean strains isolated after 2000, whereas Korean BCoV strains isolated before 2000 belonged to group GI. Mice and goats inoculated with an inactivated KBR-1 strain (isolated from this study) generated higher antibody titers (96 ± 13.49 and 73 ± 13.49, respectively) when mixed with the Montanide01 adjuvant than when mixed with the Carbopol or IMS1313 adjuvants. Viral antigens were detected in the large intestine, jejunum, and ileum of calves inoculated with inactivated KBR-1 vaccine (104.0 TCID50/mL) at 14 days of post-challenge (DPC). However, no viral antigens were detected in calves vaccinated with a higher dose of inactivated KBR-1 strain (106.0 TCID50/mL) at 14 DPC, and they had high antibody titers and stable diarrhea scores. Currently, the group GIIa is prevalent in cows in South Korea, and although further research is needed in the future, the recently isolated KBR-1 strain has potential value as a new vaccine candidate.

Evaluation of a novel multiplex qPCR method for rapid detection and quantification of pathogens associated with calf diarrhoea.

AIMS: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea. METHODS AND RESULTS: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%-103% and detection limits of 100-1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. CONCLUSION: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. SIGNIFICANCE AND IMPACT OF STUDY: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

Therapeutic potential of cristobalite in the treatment of calf diarrhea.

Calf diarrhea continues to be the major problem of calves in the neonatal period. The effect of zeolites has been increasingly studied in ruminant health in recent years. In the present study, the efficacy of cristobalite, a zeolite, in neonatal calf diarrhea was studied first time. For this purpose, twenty-five neonatal calves with diarrheas were divided into two groups, and Group 1 (n=12) received conventional treatment and Group 2 (n=13) received cristobalite (Zoosorb 10 mg/kg) orally 3 times a day in addition to conventional treatment. Escherichia coli k99 and CS31a, bovine rotavirus and bovine coronavirus were isolated from fecal samples at the beginning of the treatment, on the third day and before discharge. It was determined that the recovery period in Group 2 was 0.95 (20.6%) days shorter than in Group 1 (p⟨0.05) while no viral agents were found on the fifth day in Group 2, viral shedding continued in 4 of 5 calves in Group 1. In conclusion, the study revealed that cristobalite speeds the recovery time and possibly decreases viral shedding in neonatal calf diarrhea, demonstrating a remarkable efficiency in the treatment.

Investigation of hemogram, oxidative stress, and some inflammatory marker levels in neonatal calves with escherichia coli and coronavirus diarrhea.

Calf diarrhea is the most common disease affecting calves in the neonatal period resulting in economic losses. Although predisposing factors play a role in the etiology of the disease, in most cases, different pathogens are involved in the development of the infection. In this study, hemogram data, glutathione and malondialdehyde levels were examined to determine lipid peroxidation and glutathione levels in E. coli- and coronavirus-infected calves. Serum amyloid A and calprotectin levels were also analyzed to determine inflammatory status. The study included a total of 45 female Montofon calves aged 0-1 week, including the E. coli group (15 calves), the coronavirus group (15 calves), and the control group (15 calves). Analysis revealed that total leukocyte, neutrophil, lymphocyte, malondialdehyde, serum amyloid A, and calprotectin levels increased in the coronavirus-infected calves compared with the E. coli group and the control group. In contrast, the levels of glutathione, one of the antioxidant markers, decreased. In conclusion, the main findings related to the determination of inflammation and oxidative status were characterized by the presence of E. coli and coronavirus diarrhea, and it is suggested that future studies may be guided by the fact that inflammatory conditions are higher in viral disease than in bacterial infection.

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle.

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

Evaluation of immunochromatographic point-of-care tests for the detection of calf diarrhoea pathogens in faecal samples

We have evaluated the diagnostic performance of immunochromatographic point-of-care tests (POCT) for the detection of rotavirus, coronavirus, Escherichia (E.) coli F5, Cryptosporidium (C.) parvum, Clostridium (Cl.) perfringens and Giardia (G.) intestinalis in fresh and thawed faecal samples from calves aged up to six months with diarrhoea. We performed POCTs to detect rotavirus, coronavirus, E. coli F5, C. parvum, Cl. perfringens and G. intestinalis on fresh samples in a field study and re-evaluated the performance for C. parvum, Cl. perfringens and G. intestinalis using thawed samples. We calculated the performance based on the results of the reference methods, which were RT-qPCR for the detection of rota- and coronavirus and bacteriological culturing and PCR to detect E. coli F5 and Cl. perfringens a and ss2 toxins. C. parvum was detected by phase-contrast microscopy and G. intestinalis by immunofluorescence microscopy. We collected 177 faecal samples from diarrhoeic calves. We found good performance for the POCT targeting rotavirus (sensitivity (SE)=92.9%;specificity (SP)=95.6%) and C. parvum (SE=63.3%;SP=96.2%). For E. coli F5, the number of true positive samples (n=1) was too low to evaluate the performance. The POCT to detect coronavirus gave a poor performance (SE=3.3%;SP=96.6%) and the POCT to detect Cl. perfringens a moderate performance (SE=52.8%;SP=78.2%). G. intestinalis POCT showed a higher sensitivity to immunofluorescence microscopy in thawed than in fresh faecal samples (SE=43.9% versus SE=29.2%). There are substantial differences in diagnostic performance between the commercially available immunochromatographic POCTs. Still, POCT can make a valuable contribution to the diagnosis and prevention of calf diarrhoea.

Enteritis in calves: anamnestic, clinical, diagnostic and therapeutic evaluations with special attention to enteropathogenic rotavirus and bovine coronavirus

Introduction: Neonatal calf diarrhoea is a multifactorial disease that sometimes leads to high economic losses. It can be fatal due to dehydration and acidosis and has been one of the main causes of calf mortality. Material and methods: This retrospective study considered calves of a maximum of 35 days of age and with a diagnosed infection with rotavirus and/or bovine coronavirus. We examined the clinical records of 156 calves that were referred to the University Clinic for Ruminants in Vienna. Results Calves that had been treated with antibiotics before admission to the Clinic had a higher risk of staying longer, suggesting either that these calves had a more serious illness or that antibiotic treatment was not indicated and so therapeutic success was not achieved. Twenty-three calves died or were euthanized at the Clinic. At the time of admission, they were younger than the surviving calves and they had a lower inner body temperature and a lower base excess at the first examination. The four most common pathogens in faecal samples were rotavirus, bovine coronavirus, Cryptosporidium parvum and Escherichia coli, which were detected in 67.1%, 53.9%, 48.1% and 94.1% of the faecal samples examined. The most common co-infection was rotavirus with Cryptosporidium parvum (17 faecal samples). We inspected the four most common pathogens in more detail. There were significant correlations between bovine coronavirus and season, with the risk of suffering from bovine coronavirus 1.6 times higher in winter than in other seasons. There was also a correlation between Cryptosporidium parvum and general behaviour: the risk of being infected with Cryptosporidium parvum was 2.6 times higher in calves that were moderately to severely depressed at the first examination. There was a correlation between co-infections and mortality, with calves with a co-infection at three times higher risk of dying than calves with a mono-infection.

An Evaluation of Different Types of Peptone as Partial Substitutes for Animal-derived Serum in Vero Cell Culture.

Vero cells are one of the most frequently used cell types in virology. They can be used not only as a vehicle for the replication of viruses, but also as a model for investigating viral infectivity, cytopathology and vaccine production. There is increasing awareness of the need to limit the use of animal-derived components in cell culture media for a number of reasons, which include reducing the risk of contamination and decreasing costs related to the downstream processing of commercial products obtained via cell culture. The current study evaluates the use of protein hydrolysates (PHLs), also known as peptones, as partial substitutes for fetal bovine serum (FBS) in Vero cell culture. Eleven plant-based, two yeast-based, and three casein-based peptones were assessed, with different batches evaluated in the study. We tested the effects of three concentration ratios of FBS and peptone on Vero cell proliferation, four days after the initial cell seeding. Some of the tested peptones, when in combination with a minimal 1% level of FBS, supported cell proliferation rates equivalent to those achieved with 10% FBS. Collectively, our findings showed that plant-based peptones could represent promising options for the successful formulation of serum-reduced cell culture media for vaccine production. This is especially relevant in the context of the current COVID-19 pandemic, in view of the urgent need for SARS-CoV-2 virus production for certain types of vaccine. The current study contributes to the Three Rs principle of reduction, as well as addressing animal ethics concerns associated with FBS, by repurposing PHLs for use in cell culture.

Evaluation of diagnostic methods for the detection of Bovine Coronavirus and Rotavirus in faeces of diarrhoeic calves

The purpose of the present study was to evaluate the efficacy of Enzyme-Linked Immunosorbent Assay (ELISA), immunochromatographic (ICG), and reverse transcription-polymerase chain reaction (RT-PCR) methods for the detection of rotavirus (RV) and bovine coronavirus (BCV). Faeces samples were collected from 90 diarrhoeic calves (male and female) up to one month of age and the immune response against RV and BCV infection was assessed by using AgELISA, ICG, and RT-PCR. To determine the performance and accuracy of each diagnostic method in comparison to the diagnostic gold standard (RT-PCR) method, different statistical tests including receiver operating characteristic curve (ROC) and concordance correlation were used. Results revealed the prevalence of RV and BCV and RV+BCV according to RT-PCR were equal to 8.89 (95% CI: 6.64-10.07), 14.44 (95% CI: 11.23-6.90), and 2.22 (95% CI: 0.89-3.72), respectively. The best agreement and the highest sensitivity and specificity were obtained between the RT-PCR and AgELISA (100% and 94.3%), and also the ICG test (95% and 94.3%) was less accurate method in comparison to ELISA method for identifying RV and BCV, but a good correlation and concordance between ICG diagnostic techniques and RT-PCR were observed. To put it in a nutshell, our results demonstrate that the AgELISA is the most accurate technique in comparison to RT-PCR, however the ICG assay can help improve the speed of diagnosis RV and BCV infections in dairy field. New scientific strategies for promoting accuracy and transparency of ICG-based technique in early diagnosis of the cause of calf diarrhoea should be used. Altogether, we suggest that positive ICG samples should be tested by AgELISA or RT-PCR techniques to avoid false results in farm animals.