Feces Of Calves Research Articles

Genome characterization of a Turkish bovine rotavirus field isolate by shotgun metagenomics.

A bovine rotavirus (BRV) isolate from Kirsehir was isolated from feces of a neonatal calf with diarrhea, identified, and sequenced by shotgun sequencing. Its genotype constellation is G10-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The structural genes and the non-structural genes NSP1, NSP3, and NSP4 of the Kirsehir isolate were similar in sequence to those of BRVs identified in Turkey. However, VP2, NSP2, NSP4, and NSP5/6 showed similarity to those of rotaviruses from different animal hosts. These findings not only expand our current understanding of the diversity of rotaviruses but also contribute to our understanding of the evolution of rotaviruses at both the national and global levels and reinforce the significance of conducting further research on rotaviruses in Turkey.

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.

Systematic Surveillance of an Emerging Picornavirus among Cattle and Sheep in China.

Emerging viruses are a constant threat to human and animal health. Boosepivirus is a novel picornavirus considered a gastrointestinal pathogen and has broken out in recent years. In 2020, we identified a strain of boosepivirus NX20-1 from Chinese calf feces and performed genetic characterization and evolutionary analysis. NX20-1 was closely related to the Japanese strain Bo-12-38/2009/JPN and belonged to Boosepivirus B. We found that 64 of 603 samples (10.6%) from 20 different provinces across the country were positive for boosepivirus by reverse transcription (RT)-PCR. Further, coinfection with other diarrheal pathogens was also present in 35 of these positive samples. Importantly, we found the prevalence of boosepivirus in sheep as well, indicating that Boosepivirus can infect different domestic animals. Our data suggest that boosepivirus is a potential diarrheal pathogen, but the pathogenicity and the mechanism of pathogenesis need further study. IMPORTANCE We identified a novel picornavirus, boosepivirus, for the first time in China. Genetic evolutionary analysis revealed that NX20-1 strain was closely related to the Japanese strain Bo-12-38/2009/JPN and belonged to Boosepivirus B. In addition, we found that the virus was prevalent in China with an overall positivity rate of 10.6% (64 of 603 samples), and there was significant coinfection with other pathogens. Importantly, we found the prevalence of boosepivirus in sheep as well, suggesting that boosepivirus has a risk of spillover and can be transmitted across species.

Techno-functional characterization of fecal lactobacilli isolates of Bos indicus calves for probiotic properties.

In this study, 105 bacterial colonies were isolated from the feces of newborn healthy Bos indicus calves and 37 isolates were confirmed using morphological, biochemical tests, and genus-specific PCR as lactobacilli. 11 isolates were then short-listed for in vitro probiotic testing based on their ability to dwell under acid and bile stress. Species-level identification using 16S rRNA gene sequencing revealed that they were Ligilactobacillus salivarius. These isolates flourished in 0.4% phenol, depicting resistance in adverse conditions encountered in the gastrointestinal tract. The results of cell surface hydrophobicity were found to be 74.50% for RBL12 and 62.62% for RBL09 in hexadecane and xylene, respectively, and that of auto-aggregation was highest in RBL26 (58.92%). These isolates also produced digestive enzymes like amylase, protease, and β-galactosidase. Further assays reiterated their antimicrobial and coaggregation potential against diarrhea-causing pathogens like Escherichia coli ATCC-25922 and Salmonella arizonae ATCC-13314. Biosafety assessment revealed that none of the tested isolates were hemolytic and mucinolytic in nature. Furthermore, the antioxidant potential of the isolates was also confirmed using 1,1‑diphenyl‑2‑picrylhydrazyl (DPPH) and ferric ion-reducing antioxidant power (FRAP) assay. Along with efficient utilization of inulin, isolates showed promising adhesion ability to the HT-29 cell line. The current findings hence conclude that these Lactobacillus isolates can be exploited as animal probiotics for potentialapplication in young calves to foster gut health and immunity.

Dynamic changes in intestinal microbiota and metabolite composition of pre-weaned beef calves.

Gut microbes and their metabolites are essential for maintaining host health and production. The intestinal microflora of pre-weaned calves gradually tends to mature with growth and development and has high plasticity, but few studies have explored the dynamic changes of intestinal microbiota and metabolites in pre-weaned beef calves. In this study, we tracked the dynamics of faecal microbiota in 13 new-born calves by 16S rRNA gene sequencing and analysed changes in faecal amino acid levels using metabolomics. Calves were divided into the relatively high average daily gain group (HA) and the relatively low average daily gain group (LA) for comparison. The results demonstrated that the alpha diversity of the faecal microbiota increased with calf growth and development. The abundance of Porphyromonadaceae bacterium DJF B175 increased in the HA group, while that of Lactobacillus reuteri decreased. The results of the LEfSe analysis showed that the microbiota of faeces of HA calves at eight weeks of age was enriched with P. bacterium DJF B175, while Escherichia coli and L. reuteri were enriched in the microbiota of faeces of LA calves. Besides, the total amino acid concentration decreased significantly in the eighth week compared with that in the first week (P < 0.05). Overall, even under the same management conditions, microorganisms and their metabolites interact to play different dynamic regulatory roles. Our results provide new insights into changes in the gut microbiota and metabolites of pre-weaned calves.

First Report of OXA-48 and IMP Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Diarrheic Calves in Tunisia.

Antimicrobial resistance is one of the most serious threats to human and animal health. Evidence suggests that the overuse of antimicrobial agents in animal production has led to the emergence and dissemination of multidrug-resistant isolates. The objective of this study was to assess the rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in calf feces and to characterize their resistance genes for antibiotics like beta-lactams and colistin, but also to determine their virulence genes. Fecal samples were collected from 100 diarrheic calves in the region of Bizerte, Tunisia. After isolation, E. coli isolates were screened for antimicrobial resistance against 21 antibiotics by the disc diffusion method. Characterization of β-lactamase genes and determination of associated resistance genes were performed by polymerase chain reaction. Among 71 E. coli isolates, 26 (36.6%) strains were ESBL-producing. Most of these isolates were multidrug-resistant (92.3%) and the most prevalent beta-lactamase genes detected were blaCTX-M (n = 26), blaSHV (n = 11), and blaTEM (n = 8), whereas only 1 isolate carried the blaCMY gene. In addition, resistance to carbapenems was detected in two isolates; one of them harbored both blaOXA-48 and blaIMP genes and the other isolate carried only the blaIMP gene. Several resistance genes were identified for the first time in Tunisia from cases of diarrheic calves. Furthermore, to the best of our knowledge, this is the first report of detection and identification of carbapenem resistance genes and virulence genes from calves in North Africa. A high occurrence of antimicrobial resistance of E. coli recovered from fecal samples of calves with diarrhea was observed, highlighting the need for prudent use of antimicrobial agents in veterinary medicine to decrease the incidence of multidrug-resistant bacteria for both animals and humans.

The probiotic and immunomodulation effects of Limosilactobacillus reuteri RGW1 isolated from calf feces.

Limosilactobacillus reuteri is a gut symbiont with multiple remarkable beneficial effects on host health, and members of L. reuteri are valuable probiotic agents. However, L. reuteri showed obvious host specificity. In our study, a novel L. reuteri RGW1 was isolated from feces of healthy calves, and its potential as a probiotic candidate were assessed, by combining in vitro, in vivo experiments and genomic analysis. RGW1 was sensitive to all the antibiotics tested, and it did not contain any virulence factor-coding genes. This isolate showed good tolerance to acid (pH 3.0), 0.3% bile salt, and simulated gastric fluid. Moreover, this isolate showed a high hydrophobicity index (73.7 ± 4.6%) and was able to adhere to Caco-2 cells, and antagonize Escherichia coli F5. Treatment of LPS-induced mice with RGW1 elevated TGF-β and IL-10 levels, while RGW1 cell-free supernatant (RCS) decreased TNF-α levels in the sera. Both RGW1 and RCS increased the villus height and villus height/crypt depth ratio of colon. Genomic analysis revealed the mechanism of the probiotic properties described above, and identified the capacity of RGW1 to biosynthesize L-lysine, folate, cobalamin and reuterin de novo. Our study demonstrated the novel bovine origin L. reuteri RGW1 had multiple probiotic characteristics and immunomodulation effects, and provided a deeper understanding of the relationship between these probiotic properties and genetic features.

Transcriptional changes detected in fecal RNA from neonatal dairy calves of different breeds following gastrointestinal disease of varying severity.

Gastrointestinal (GI) disease is a major health concern in preweaned dairy calves. The objective of this fixed cohort study was to use RNA isolated from preweaned Holstein and Jersey heifer calf feces to study the molecular adaptations to variable clinical GI disease. The study was conducted on a commercial calf ranch in the western U.S. Enrolled calves were assessed twice daily for variations in demeanor, milk intake, and hydration. Fecal consistency scores were recorded at enrollment (day 1), and on the day (day 10) that a fecal sample was collected for differential gene expression (DGE). Calves with diarrhea on either day were classified as having either uncomplicated, localized GI disease (scours), or systemic GI disease (systemic enteritis). Eighty-four calves' fecal RNA was evaluated for DGE, of which 33 calves (n = 20 Holstein; n = 13 Jersey) were consistently healthy. The remaining 51 calves (n = 23 Holstein; n = 28 Jersey) experienced varying severity of GI disease during the sampling window. Genes of interest were related to the inflammatory response (i.e., IFNG, NFKB1, NOD2, TLR2, and TLR4) and cell membrane or cytoplasmic transport (i.e., AQP3, FABP2, KRT8 and SLC5A1). Breed-specific findings indicated that AQP3, IFNG, and TLR4 were upregulated in Holsteins with systemic enteritis, whereas KRT8 was downregulated in systemically affected Jerseys. Holsteins did not appear affected by scours aside from a tendency for DGE of toll-like receptors (TLRs) on the day of diarrhea. However, Jersey calves consistently demonstrated a tendency to upregulate IFNG, NFKB1, and TLR4 when affected with either scours or systemic enteritis. These findings were more pronounced in systemically affected Jersey calves and were observed as a delayed response to both scours and systemic enteritis. These findings support previous observations suggesting that Holstein calves may be better equipped than Jersey calves to rapidly fight pathogen invasion.

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.

Abundance of selected bacterial groups in healthy calves and calves developing diarrhea during the first week of life: Are there differences before the manifestation of clinical symptoms?

Diarrhea is still the most common and economically most significant disease of newborn calves. Analysis of the development of selected bacterial groups in the feces of neonatal calves and its significance regarding diarrhea. A total of 150 newborn Simmental calves reared in 13 Bavarian farms were included in the study. Fecal samples of calves taken at 0/6/12/24/48/72/168 hours (h) since birth were analyzed qualitatively and quantitatively for aerobic and anaerobic bacteria, such as Enterobacteriaceae, E. coli, enterococci, and lactobacilli, using cultural, biochemical, and molecular-biological methods. Concurrently, the health status of the animals was recorded. The bacterial levels of healthy and diarrheic animals were compared using statistical methods. In addition, feces samples from calves that developed diarrhea were examined by ELISA for the presence of rotaviruses, coronaviruses, E. coli F5, and Cryptosporidium (Cr.) parvum. Fifty-seven out of 150 calves (37.3 %) that were examined developed diarrhea within the first week of life. In the feces of calves with diarrhea on day 1 of life, the levels of aerobes, Enterobacteriaceae, and E. coli were significantly increased (p < 0.05), while no significant differences in enterococci and lactobacilli were found. In animals with the onset of diarrhea on day 2 after birth, the load of lactobacilli was significantly reduced up to 24 h before the manifestation of clinical symptoms compared to healthy calves. For enterococci, this was only the case on the day of the onset of diarrhea. In addition, the ratios of aerobic and anaerobic bacteria, Enterobacteriaceae or E. coli to lactobacilli, of calves with diarrhea starting on day 2 after birth are significantly higher than those of healthy calves. The detection frequency of specific pathogens in diarrheic calves increased over the first week of life. The results suggest that the incidence of neonatal diarrhea in calves is favored by low levels of lactobacilli in the feces. From this, the hypothesis can be derived that, in addition to an optimal supply of colostrum, the earliest possible administration of lactobacilli might reduce neonatal diarrhea in calves. However, this must be verified in a subsequent feeding experiment.

Stall Sanitation Associated with Cryptosporidium sp. Infection on Calf and Cattle Farmer

Increasing the cattle population can increase the amount of the waste. Poor management of cage sanitation has the risk of causing a decrease in the health conditions of cattle farmers, such as: diarrhea, nausea, vomiting, and itching, as many as 50% of cattle with diarrhea are infected with Cryptosporidium sp. The highest infection of Cryptosporidium sp. in cattle found in calves. The calf can excrete 1×1010 oocysts of Cryptosporidium sp. per day. Cryptosporidium sp. also has the potential to cause zoonotic infections that are transmitted from animals to humans. The study aims was to determine the relationship between stall sanitation and Cryptosporidium sp. to calf and cattle farmer. The type of this research was analytic observational with a cross sectional approach conducted in Jatian Village, Pakusari, Jember during October 2021 - March 2022. The primary data was obtained through interviews and observations. The feces of calves and cattle farmer were collected and examined using sedimentation method and modified Ziehl-Neelsen (MZN) staining method. The statistical test used is the chi-square test. The results showed that the prevalence of Cryptosporidium sp. in calves by 14,3% and on cattle farmer by 4,8%. The bivariate analysis showed that that stall location and waste handling were associated with Cryptosporidium for calf (p≤0.05), and there were no variables that were significantly related to infection in cattle farmers (p&gt;0,05). Cryptosporidium sp. in calves and humans can be caused by several factors, such as environmental factors and hosts. Preventive measures such as maintaining a clean environment including cowshed sanitation and personal hygiene need to be taken to reduce the risk of Cryptosporidium infection from calves to humans, and vice versa.

Effects of fecal microbiota transplantation from yaks on weaning diarrhea, fecal microbiota composition, microbial network structure and functional pathways in Chinese Holstein calves.

This study was conducted to investigate the effect of fecal microbiota transplantation (FMT) from yaks on weaning diarrhea, fecal microbiota composition, microbial network structure and functional pathways in Chinese Holstein Calves. In this study, 50 calves were randomly divided into five groups of 10 each: NC group (no supplementation), Control group (normal saline), low concentration FMT group (LFMT, 1 × 108 CFU/ml), high concentration FMT group (HMFT, 1 × 109 CFU/ml), and sterilized FMT group (SMFT, sterilized bacterial solution). The test lasted for 30 days. We found that FMT reduced the incidence of diarrhea in weaned calves, and the anti-diarrhea effect of LFMT was stronger than those of HFMT and SFMT. Calf feces were collected by rectal palpation on days 5, 10, 15, and 20 post-weaning, and high-throughput sequencing of bacterial 16S rRNA and fungal internal transcribed spacer region of fecal microbiota was performed. We observed that the richness and diversity of bacterial microbiota in the LFMT, HFMT, and SFMT groups were higher than those in the NC and Control groups at day 20 after weaning. The treatment had a significant effect on bacterial richness (p < 0.05), but not on fungal diversity or richness. The analysis of gut microbiome showed that Firmicutes and Bacteroides were the main bacterial phyla in the feces of weaned calves, and norank_ f Muribaculaceae, UCG-005, Rikenellaceae_RC9_gut_group, Bacteroides, and Blautia were the main genera. Ascomycota and Basidiomycota were the main fungal phyla. Compared to abundance parameters in the Control and NC groups, relative abundances of Firmicutes in the FMT groups increased at different time points after weaning. The relative abundance of Blautia and Lactobacillus in the LFMT group increased significantly after weaning. In addition, abundances of Ruminococcus and Romboutsia, which produce short-chain fatty acids, were also increased in different FMT groups. FMT significantly increased the relative abundance of beneficial bacteria, enhanced the complexity of the fecal microbial network, and promoted important metabolic and cellular processes in weaned calves. In conclusion, our study provides a reference and theoretical basis for FMT to prevent calf weaning diarrhea and other intestinal diseases in ruminants.

Determination of an RT-qPCR viral load cutoff point for the etiologic diagnosis of rotavirus A diarrhea in neonate dairy calves.

Rotavirus A (RVA) is amongst the most widespread causes of neonatal calf diarrhea. Because subclinical infections are common, the diagnosis of RVA-induced diarrhea cannot rely solely on molecular viral detection. However, RT-qPCR allows for quantification of RVA shedding in feces, which can be correlated with clinical disease. Here, we determine an optimal cutoff of rotaviral load quantified by RT-qPCR to predict RVA causality in diarrheic neonate calves, using RVA antigen-capture ELISA as reference test. Feces from 328 diarrheic (n = 175) and non-diarrheic (n = 153), <30-day-old dairy calves that had been tested by ELISA and tested positive by RT-qPCR were included. Of 82/328 (25.0%) ELISA-positive calves, 53/175 (30.3%) were diarrheic, whereas 124/153 (81.0%) non-diarrheic calves tested negative by ELISA. The median log10 viral load was significantly higher in diarrheic vs. non-diarrheic and ELISA-positive vs. -negative calves, indicating a higher viral load in diarrheic and ELISA-positive calves. A receiver operating characteristic (ROC) analysis was conducted using the viral loads of the 175 diarrheic calves that had tested either positive (n = 53, cases) or negative (n = 122, controls) by ELISA. The optimal log10 viral load cutoff that predicted RVA causality in diarrheic calves was 9.171. A bootstrapping procedure was performed to assess the out-of-bag performance of this cutoff point, resulting in sensitivity = 0.812, specificity = 0.886, area under the curve = 0.922, and positive and negative diagnostic likelihood ratios of 11.184 and 0.142, respectively. The diagnostic accuracy of the cutoff was excellent to outstanding. This information will help in the interpretation of RVA RT-qPCR results in feces of diarrheic calves submitted for laboratory testing.

Silver nanoparticle effect on Salmonella enterica isolated from Northern West Egypt food, poultry, and calves

A total no. of 65 Salmonella enterica isolates recovered from food samples, feces of diarrheic calves, poultry, and hospital patient in large five cities at Northern West Egypt were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. The 65 Salmonella enterica isolates had the invA gene were grouped into 11 Salmonella enterica serovars with dominance of S. Enteritidis and S. Kentucky serovars. Their resistance pattern were characterized by using 18 antibiotics from different classes. Approximately 80% of the isolates were multidrug resistant (MDR). Enterobacterial repetitive intergenic consequences polymerase chain reaction (ERIC-PCR) typing of 7 strains of S. Enteritidis showed 5 clusters with dissimilarity 25%. S. Enteritidis clusters in 2 main groups A and B. Group A have 2 human strain (HE2 and HE3) and one food origin (FE7) with a similarity 99%. Group B divided into B1 (FE2) and B2 (FE3) with a similarity ratio ≥ 93%, while ERIC-PCR analysis of 5 strains of S. Kentucky revealed 4 ERIC types, clustered in 2 main groups A and B with similarity 75%. We studied the effect of silver nanoparticles (Ag-NPs) on 10 antibiotic resistant strains of S. Enteritidis and S. Kentucky. The broth microdilution minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected. Evaluation of the affection using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed different ratios of Ag-NPs and microorganism as well as at different contact time ended finally with morphological alteration of the bacteria. We submitted new method in vivo to explore the activity of nanosilver in chicken.Key points• Importance of ERIC-PCR to determine the relatedness between Salmonella isolates.• Effect of silver nanoparticles to confront the antibacterial resistance.• Studying the effect of silver nanoparticles in vivo on infected chicken with Salmonella.

High Fecal Prevalence of mcr-Positive Escherichia coli in Veal Calves at Slaughter in France.

The aim of this study was to determine the percentage of healthy veal calves carrying mcr-positive E. coli strains at the time of slaughter in France. Fecal samples were selectively screened for mcr-positive E. coli isolates using media supplemented with colistin. Screening for mcr genes was also carried out in E. coli isolates resistant to critically important antimicrobials used in human medicine recovered from the same fecal samples. Overall, 28 (16.5%) out of the 170 veal calves tested carried mcr-positive E. coli. As some calves carried several non-redundant mcr-positive strains, 41 mcr-positive E. coli were recovered. Thirty-one and seven strains were positive for mcr-1 and mcr-3 genes, respectively, while no strain was positive for the mcr-2 gene. Co-carriage of mcr-1 and mcr-3 was identified in three strains. All mcr-positive E. coli isolates, except one, were multidrug-resistant, with 56.1% being ciprofloxacin-resistant and 31.7% harboring blaCTX-M genes. All mcr-3-positive E. coli carried blaCTX-M genes, mainly blaCTX-M-55. This study highlights the high prevalence of mcr-positive E. coli strains in feces of veal calves at the time of slaughter. It also points out the multidrug (including ciprofloxacin) resistance of such strains and the co-occurrence of mcr-3 genes with blaCTX-M-55 genes.

Shift of dietary carbohydrate source from milk to various solid feeds reshapes the rumen and fecal microbiome in calves

The transition from milk to solid diets drastically impacts the gut microbiome of calves. We explored the microbial communities of ruminal fluid and feces of Holstein calves when fed milk on d 7 of life, and when fed solid feeds based on either medium- or high-quality hay with or without concentrate inclusion (70% in fresh matter) on d 91. Ruminal fluid and feces had distinct microbial compositions already on d 7, showing that niche specialization in early-life gut is rather diet-independent. Changes between d 7 and d 91 were accompanied by a general increase in microbial diversity. Solid diets differed largely in their carbohydrate composition, being reflected in major changes on d 91, whereby concentrate inclusion was the main driver for differences among groups and strongly decreased microbial diversity in both matrices. Fecal enterotyping revealed two clusters: concentrate-supplemented animals had an enterotype prevalent in Prevotella, Succinivibrio and Anaerovibrio, whereas the enterotype of animals without concentrate was dominated by fibrolytic Ruminococcaceae. Hay quality also affected microbial composition and, compared to medium-quality, high-quality hay reduced alpha-diversity metrics. Concluding, our study revealed that concentrate inclusion, more than hay quality, dictates the establishment of niche-specific, microbial communities in the rumen and feces of calves.

Evaluation of diagnostic methods for the Detection of Bovine Coronavirus and Rotavirus in feces of diarrheic 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). Feces 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 ELISA (Ag and Ab), 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), concordance correlation were used. Results shown 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 then AbELISA (100% and 94.3%) was the second-best test and also the ICG test (95% and 94.3%) was less accurate method in comparison to ELISA methods 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 suggest that the ICG assay may improve the ability to diagnose calves RV and BCV infections accurately and quickly. Promoting rapid IGG kit with higher accuracy in early diagnosis of the cause of diarrhoea plays an important role in its therapeutic regimes, management protocols, and control procedures, but ELISA is preferred due to more precise results.

Effects of Pathogenic Escherichia coli Infection on the Flora Composition, Function, and Content of Short-Chain Fatty Acids in Calf Feces.

Simple SummaryCalf diarrhea caused by bacteria usually begins to occur 2–3 d after birth. The main pathogenic bacteria are Escherichia coli, Campylobacter, Salmonella, and Clostridium perfringens. Pathogenic E. coli is the main bacterium that causes diarrhea in calves. It invades the body of calves and releases enteric toxins that cause diarrhea. The incidence of calf diarrhea caused by pathogenic E. coli O1 can reach 60–70%, even as high as 90–100% in large-scale calf farms, and the mortality rate can reach over 50%. Therefore, the prevention and treatment of calf diarrhea are extremely important. We established a calf diarrhea model by artificially intervening in the colonization of intestinal microbial flora and studied the influence of pathogenic E. coli on the composition, function, and short-chain fatty acid content of calf feces. Our research shows that pathogenic E. coli O1 can significantly reduce the abundance and diversity of calf intestinal microflora, increase harmful bacteria, reduce the colonization of beneficial bacteria, and reduce the short-chain fatty acid content in feces.Calf diarrhea caused by pathogenic Escherichia coli is a major cause of death in calves, with a mortality rate of over 50%. It is crucial to understand the pathogenesis and development of calf diarrhea for its prevention and treatment. We aimed to study the effect of pathogenic E. coli on the flora composition, function, and short-chain fatty acid (SCFA) content of calf feces using a calf diarrhea model. Sixty-four newborn Holstein calves (40–43 kg) were divided into a normal group (NG; n = 32) and a test group (TG; n = 32). At the beginning of the experiment, the TG were orally administered pathogenic E. coli O1 (2.5 × 1011 CFU/mL, 100 mL) to establish a calf diarrhea model, and the NG were orally administered the same amount of physiological saline solution. The calves of the two groups were subjected to the same feeding and management. Fresh feces samples were collected at different time points and subjected to 16S rRNA high-throughput sequencing and gas chromatography–mass spectrometry to determine the fecal microbial composition and SCFA content. Pathogenic E. coli O1 significantly altered microbiotas composition in the feces of calves, increasing the relative abundance of Proteobacteria and decreasing that of Firmicutes. It also led to a significant increase in the relative abundance of Escherichia-Shigella and a decrease in Lactobacillus, as well as significantly decreased SCFA content. Therefore, we postulate that pathogenic E. coli induces calf diarrhea by causing intestinal florae imbalance and reducing the content of SCFA.

Management of calves in commercial dairy farms in Mecklenburg-Western Pomerania, Germany and its impact on calf mortality and prevalence of rotavirus and Cryptosporidium parvum infections in pre-weaned calves

In a cross-sectional study, impact of management in dairy farms on calf mortality rates and prevalence of rotavirus and Cryptosporidium parvum in feces of calves was investigated. Sixty-two commercial dairy herds in Mecklenburg-Western Pomerania, Germany, were stratified selected in 2019. We performed in-person interviews and fecal specimens in samples of all-female calves of age 7 up to 21 days. Management data were documented on farm level. A Multiscreen Ag-ELISA was performed to determine rotavirus and Cryptosporidium parvum. Associations between two calf mortality rates, detection of C. parvum and rotavirus, and predictors were examined with GLM models. In farms with routine vaccination against respiratory diseases, 31-days mortality rate was 4.2% +/-1.26 compared to 7.6% +/-0.97 (p = 0.040) on non-vaccinating farms. Six-months mortality was lower in farms that continued feeding milk to calves during periods of diarrhea compared to farms that did not (6.9% +/-0.8 vs. 12.4% +/-2.3). In case of a routine shifting of calves from the calving box into calf boxes less C. parvum was detected compared to an individual moving of calves (33.3% +/-2.6 vs. 19.6% +/-5.3; p = 0.024). Our model confirms a positive association between occurrence of aqueous feces and frequency of detection of C. parvum (45.4% +/-23.6 vs. 21.4% +/-18.7; p < 0.001). Frequency of detection of rotavirus was lower in farms that reported a defined amount of applicated colostrum per calf than in farms that presented a range of colostrum instead of a defined amount. This study indicates the potential for mitigation of risk factors for mortality in calves.

Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.