Calf Fecal Samples Research Articles

Bovine fecal biomarkers of intestinal inflammatory process: Calprotectin and lactoferrin, a comparative study.

Fecal lactoferrin and fecal calprotectin have been proposed as biomarkers of intestinal inflammation in several animal species. The main objectives of this work were to validate an analytical procedure for the measurement of lactoferrin in calf feces, to study the correlation between lactoferrin and calprotectin concentrations, and to evaluate the influence of fecal water content in the determination of these proteins. This knowledge is essential for effectively using these biomarkers in young calves exposed to inflammatory gastrointestinal diseases.Seventy-eight male Holstein dairy calves between two and three weeks of age were included in the study. Lactoferrin was determined with a bovine milk lactoferrin ELISA kit and calprotectin was measured using a human immunoturbidimetric method previously validated in bovine feces. Analytical validation of the lactoferrin assay achieved good results, with intra and inter assay CV<10%, recovery rates between 80 and 120%, and optimal linearity under dilution. A robust correlation was observed between fecal calprotectin concentrations in dry and wet feces (r=0.903), while a moderate correlation was observed for fecal lactoferrin concentrations (r=0.648). Correlation between both biomarkers was moderate in fresh feces (r=0.514) as well as in dry feces (r=0.561). In conclusion, the lactoferrin ELISA kit is valid for its use with calf fecal samples, both biomarkers showed a moderate correlation between them, and fecal lactoferrin concentration is more influenced by feces moisture than fecal calprotectin concentration.

Residues of dry cow antimicrobials in colostrum and extended-spectrum and AmpC β-lactamaseproducing Escherichia coli in feces from young dairy calves.

The duration of excretion of dry cow antimicrobials is hard to predict and therefore the level of antimicrobial residues in colostrum can differ considerably between cows. The feeding of colostrum with antimicrobial residues to newborn calves may lead to antimicrobial resistance in calves. The aim of this study was to quantify the prevalence and residue level of dry cow antimicrobials in colostrum from dairy cows. Additionally, the association between antimicrobial residues in colostrum and the prevalence of extended spectrum and AmpC β-lactamaseproducing Escherichia coli (ESBL/AmpC-EC) in colostrum and in the feces from young dairy calves was evaluated. The work described in this paper consists of 3 studies. In the first study, in 75 of 118 colostrum samples antimicrobial residues were found, in 50 of 88 β-lactam treated cows, and in 25 of 30 cows treated with a combination of an aminoglycoside and a β-lactam. In cows that had been dried off with a combination of an aminoglycoside and a β-lactam, in significantly more samples antimicrobial residues were found. In 38 of the colostrum samples tested, antimicrobial concentrations (mainly cloxacillin) exceeded the Maximum Residue Limit. The prevalence of antimicrobial positive colostrum samples presented was much higher than earlier described. In the second study 28 dairy cows from 8 farms were dried off with either cloxacillin or a combination of neomycin and benzylpenicillin. Because in study 1 the tendency of a negative association was found between the use of teat sealants and the presence of antimicrobials in colostrum, it was hypothesized that with the striping of the teat sealant much of the antimicrobial residues was removed. This hypothesis could not be confirmed by comparing antimicrobial concentrations in the first streams of the first milking and the remainder of that milking. The 22 clocaxilin treated cows were followed up for 6 subsequent milkings, during which a decrease in the concentration of antimicrobial residues was found. In the third study, 87 cows from 10 herds were included of which 58 cows were dried off with cloxacillin while 29 cows were dried off without antimicrobials. Cows were compared with respect to antimicrobial residues in the first 5 milkings after calving and the occurrence of ESBL/AmpC-EC in the feces from their calves, collected at d 1, d 7 and d 14 of life. Significantly higher percentages of ESBL/AmpC-EC positive samples were found in feces samples collected at d 7 and d 14 compared with the d 1 samples. No significant differences were found in the prevalence of ESBL/AmpC-EC positive fecal samples between calves born from cows that had been dried off without antimicrobials and those that had been dried off with cloxacillin. Given the fact that cloxacillin does not select for ESBL/AmpC-EC, this was not unexpected. This study showed that colostrum from cows dried off with antimicrobials often contains antimicrobial residues. Although no effect of these residues on the prevalence of ESBL/AmpC-EC in fecal samples of calves was found in this study, we recommend to minimize antimicrobial use at drying off and to respect withdrawal recommendations.

Vertical transfer of gut microbiota from dam to neonate calf in the early of life

The aim of this study was to investigate the vertical transfer of microbiota from dams to the offspring. We studied a pair of 20 dams and its offspring. Maternal sources (colostrum, feces and vaginal secretion) and newborn fecal samples were analyzed using 16S rDNA amplicon sequencing on days 1, 3, 7, 14 and 28. Overall, newborns were maintained healthy and did not receive antimicrobial therapy. The Source Tracker analysis indicated that the newborn fecal microbiota was similar to colostrum and vaginal secretion from day 1 up to 7. However, an unknown source (probably from the environment) showed a gradual increase in its similarity with fecal samples from calves measured from day 3 to 28. The most abundant bacteria groups on meconium (day 1) and calf fecal samples on day 3 were Escherichia-Shigella and Clostridium, respectively. On day 7, the predominant genus were Bifidobacterium and Lactobacillus, while Fusobacterium was the most abundant genus on day 14, coinciding with the diarrhea peak. Faecalibacterium showed a gradual increase throughout the neonatal period. Maternal sources contribute to the neonatal microbiota, however other unknown sources (probably environment) had a strong influence on development of the gut microbiota later in the neonate period.

First detection of Cupriavidus gilardii in a bovine neonatal diarrhea outbreak

BackgroundCupriavidus 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.Case presentationThis 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.ConclusionThis 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 first complete genome sequence and genetic evolution analysis of bovine norovirus in Xinjiang, China

Abstract Introduction Viruses are among the main pathogens causing diarrhoea in calves. The current study found that bovine norovirus (BNoV) is one of the principal viruses causing diarrhoea in calves in Xinjiang, China. Material and Methods A total of 974 calf faecal samples from six regions in Xinjiang were tested for BNoV using reverse-transcriptase PCR. The genomic characteristics of BNoV and the genetic evolution of the VP1 gene, protein three-dimensional structure characteristics and amino acid variation were analysed using bioinformatics methods. Results Epidemiological survey results showed that the infection rate of BNoV was 19.82%, and all samples tested positive in five regions. The results of the genetic evolution analysis showed that BNoV strains from Tacheng of northern Xinjiang and Kashgar of southern Xinjiang both belonged to the GIII.2 genotype of BNoV but were not on the same cluster of evolutionary branches. Additionally, the amino acid variation of the VP1 protein was not observed to significantly affect its spatial structure. Conclusion This study is the first to report the genetic characteristics of the BNoV complete genome sequence in Xinjiang and provides a scientific basis for BNoV vaccine development and pathogenesis research.

Antimicrobial resistance and public and animal health risks associated with pathogenic Escherichia coli isolated from calves

We aimed to determine the antimicrobial susceptibility profile of pathogenic Escherichia coli strains isolated from fecal samples of calves and buffalo calves (2008–2013), in Minas Gerais, Brazil, as well as the frequency of O157 gene and strains carrying extended-spectrum beta-lactamases (ESBL) and mobile colistin resistance (mcr) genes. E. coli strains (n=518) were tested for susceptibility against ten antimicrobials. Tetracycline was the antimicrobial with the highest resistance rate (382/518), followed by ampicillin (321/518), sulfamethoxazole/trimethoprim (312/518), chloramphenicol (192/518), gentamicin (126/518), ciprofloxacin (148/518), cefazolin (89/518), colistin (54/518) and cefoxitin (34/518). Multidrug resistance (MDR) was observed in 381/518 isolates. No strain harbored mcr or O157 genes, whereas 19/99 were ESBL positive. The most prevalent pathotype and phylogroup were STEC and B1, respectively. Age, EHEC pathotype and resistance to aminoglycoside and cephem were significantly associated with MDR in the multivariate model. Overall, E. coli strains showed high rates of resistance to penicillin, tetracyclines and folate inhibitors, in addition to an alarming rate of MDR and ESBL-producing strains.

Effects of a commercial feed additive used for prophylactic purposes on health and blood parameters in neonatal calves.

Neonatal calf diarrhea is commonly observed worldwide and leads to significant economic losses. The objective of this study was to investigate the effect of a feed additive supplemented with milk on the incidence of diarrhea days with scours, fecal score, average daily gain, fecal bacterial concentration, and blood parameters in neonatal calves. Forty Holstein-Friesian crossbreed (HF x Montbeliarde) calves were divided into control (C) and experimental (E) groups, each containing 20 calves. The study was conducted over a period of 10 days. The control group was fed whole milk twice daily. The experimental group was fed the same whole milk supplemented with feed additive once in the morning at 100 g/calf/day. At the end of the study, it was determined that the feed additive in the milk affected fecal score, blood pH, pCO2, cHCO3 -, K+, cHgb, and plasma glucose levels (p<0.05), but did not affect the incidence of diarrhea, average daily gain, days with scours, and other blood parameters (p>0.05). Rotavirus was the main pathogen found in the fecal samples of calves with diarrhea. Higher concentrations of Escherichia coli, Salmonella spp., and Clostridium perfringens were detected in the feces of experimental calves (p<0.001). The findings indicate that this feed additive improves fecal score, some blood parameters, and fecal concentration of pathogenic bacteria in feces. Additionally, it shows a favorable effect on both diarrhea incidence and the days with scours, suggesting its potential efficacy in preventing diarrhea in neonatal calves. Preventing diarrhea during this crucial stage of life can contribute to the health and productivity of animals and play a role in preventing antibiotic resistance by reducing reliance on antibiotics.

Prevalence of antimicrobial resistance in fecal Escherichia coli and Enterococcus spp. isolates from beef cow-calf operations in northern California and associations with farm practices.

Antimicrobials are necessary for the treatment of bacterial infections in animals, but increased antimicrobial resistance (AMR) is becoming a concern for veterinarians and livestock producers. This cross-sectional study was conducted on cow-calf operations in northern California to assess prevalence of AMR in Escherichia coli and Enterococcus spp. shed in feces of beef cattle of different life stages, breeds, and past antimicrobial exposures and to evaluate if any significant factors could be identified that are associated with AMR status of the isolates. A total of 244 E. coli and 238 Enterococcus isolates were obtained from cow and calf fecal samples, tested for susceptibility to 19 antimicrobials, and classified as resistant or non-susceptible to the antimicrobials for which breakpoints were available. For E. coli, percent of resistant isolates by antimicrobial were as follows: ampicillin 100% (244/244), sulfadimethoxine 25.4% (62/244), trimethoprim-sulfamethoxazole 4.9% (12/244), and ceftiofur 0.4% (1/244) while percent of non-susceptible isolates by antimicrobial were: tetracycline 13.1% (32/244), and florfenicol 19.3% (47/244). For Enterococcus spp., percent of resistant isolates by antimicrobial were as follows: ampicillin 0.4% (1/238) while percent of non-susceptible isolates by antimicrobial were tetracycline 12.6% (30/238) and penicillin 1.7% (4/238). No animal level or farm level management practices, including antimicrobial exposures, were significantly associated with differences in isolate resistant or non-susceptible status for either E. coli or Enterococcus isolates. This is contrary to the suggestion that administration of antibiotics is solely responsible for development of AMR in exposed bacteria and demonstrates that there are other factors involved, either not captured in this study or not currently well understood. In addition, the overall use of antimicrobials in this cow-calf study was lower than other sectors of the livestock industry. Limited information is available on cow-calf AMR from fecal bacteria, and the results of this study serve as a reference for future studies to support a better understanding and estimation of drivers and trends for AMR in cow-calf operations.

Potential Zoonotic Transmission of Giardia duodenalis between Children and Calves in Bangladesh

Giardia duodenalis is a zoonotic protozoan parasite that causes gastrointestinal illness in humans and livestock. We studied the genetic diversity of G. duodenalis in children and calves from Bangladesh to determine its zoonotic potential. Fecal samples collected from children (299) and calves (699) were screened with nested PCR with primers targeting the ssu rRNA gene for G. duodenalis. Positive samples were further multilocus genotyped using the β-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) genes. The overall infection rate of G. duodenalis was 21.1% (63/299) in children and 5.7% (40/699) in calves. There were no significant differences in infection with G. duodenalis among age groups, sex, and study areas in children and calves. Multilocus genotyping (MLG) of human G. duodenalis identified zoonotic assemblages A (34.0%, 18/53) and B (50.9%, 27/53) and a so-called ruminant-specific assemblage E (11.3%, 6/53), as well as two mixed assemblages, B/D (1/53) and B/E (1/53). Assemblage E predominated in calves (82.3%, 28/34), followed by assemblages A (11.8%, 4/34) and B (5.9%, 2/34). Overall, zoonotic assemblages A, B, and E were found in 6.0% (18/299), 9.0% (27/299), and 2.0% (6/299) of the children’s stool samples, respectively, and 0.6% (4/699), 0.3% (2/699), and 4.0% (28/699) of the calf fecal samples, respectively. Although there was a difference in the distribution of subassemblages in humans (mostly AII) and calves (mostly AI), the zoonotic assemblages A, B, and E present in both children and calves suggest the potential for zoonotic transmission of G. duodenalis. This molecular study highlights the fact that G. duodenalis infections were common in the study areas, with potential zoonotic transmission between children and calves, implying that cattle might play a role in G. duodenalis zoonotic transmission.

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.

Phenotypic and Genotypic Assessment of Antimicrobial Resistance in Escherichia coli from Australian Cattle Populations at Slaughter

Australia relies on periodic antimicrobial resistance (AMR) surveys to determine trends and changes in AMR in animal production systems. This study is a follow-up to a survey of Escherichia coli from healthy cattle at slaughter conducted in 2013, which provided baseline data on AMR prevalence across cattle groups and production practices. In this study, 591 beef cattle, 194 dairy cattle, and 216 veal calf fecal samples were collected from 25 beef and veal processing establishments in Australia, representing approximately 77% of total export volume. A total of 969 matrix-assisted laser desorption-ionization results confirmed commensal E. coli isolates from 574 beef cattle, 186 dairy cattle, and 209 veal calves were recovered, and antimicrobial susceptibility testing was carried out by microbroth dilution to 16 drugs from 10 classes interpreted against epidemiological cutoff breakpoints. Overall, a high proportion of E. coli isolates (83.8%) were wild type for all antimicrobials assessed. In addition, isolates that were non-wild type (NWT) for three or more classes of antimicrobial did not exceed 4% for any of the cattle groups. The prevalence of E. coli that were NWT for antimicrobials that are critical or of high importance to human health was very low, with 1.4% of all isolates tested determined to be NWT for fluoroquinolones, third-generation cephalosporins, or polymyxins. Genomic analysis of NWT isolates identified one beef cattle isolate (ST-10) harboring blaCMY-2 and a dairy isolate (ST-58) and two veal calf isolates (ST-58 and ST-394) that had qnrS1, which confer resistance to extended-spectrum cephalosporins and fluoroquinolones, respectively. The low levels of AMR reported in this study confirm previous Australian studies, which indicated that there is minimal evidence that specific production practices lead to widespread disproportionate development of NWT isolates.

Virulome and genome analyses identify associations between antimicrobial resistance genes and virulence factors in highly drug-resistant Escherichia coli isolated from veal calves.

Food animals are known reservoirs of multidrug-resistant (MDR) Escherichia coli, but information regarding the factors influencing colonization by these organisms is lacking. Here we report the genomic analysis of 66 MDR E. coli isolates from non-redundant veal calf fecal samples. Genes conferring resistance to aminoglycosides, β-lactams, sulfonamides, and tetracyclines were the most frequent antimicrobial resistance genes (ARGs) detected and included those that confer resistance to clinically significant antibiotics (blaCMY-2, blaCTX-M, mph(A), erm(B), aac(6’)Ib-cr, and qnrS1). Co-occurrence analyses indicated that multiple ARGs significantly co-occurred with each other, and with metal and biocide resistance genes (MRGs and BRGs). Genomic analysis also indicated that the MDR E. coli isolated from veal calves were highly diverse. The most frequently detected genotype was phylogroup A-ST Cplx 10. A high percentage of isolates (50%) were identified as sequence types that are the causative agents of extra-intestinal infections (ExPECs), such as ST69, ST410, ST117, ST88, ST617, ST648, ST10, ST58, and ST167, and an appreciable number of these isolates encoded virulence factors involved in the colonization and infection of the human urinary tract. There was a significant difference in the presence of multiple accessory virulence factors (VFs) between MDR and susceptible strains. VFs associated with enterohemorrhagic infections, such as stx, tir, and eae, were more likely to be harbored by antimicrobial-susceptible strains, while factors associated with extraintestinal infections such as the sit system, aerobactin, and pap fimbriae genes were more likely to be encoded in resistant strains. A comparative analysis of SNPs between strains indicated that several closely related strains were recovered from animals on different farms indicating the potential for resistant strains to circulate among farms. These results indicate that veal calves are a reservoir for a diverse group of MDR E. coli that harbor various resistance genes and virulence factors associated with human infections. Evidence of co-occurrence of ARGs with MRGs, BRGs, and iron-scavenging genes (sit and aerobactin) may lead to management strategies for reducing colonization of resistant bacteria in the calf gut.

A comparison of assays for the detection of Cryptosporidium parvum in the feces of scouring calves.

Cryptosporidium parvum is a zoonotic, protozoan parasite that causes potentially life-threatening diarrhea in the host and can be transmitted via the fecal-oral route. C. parvum can infect cattle and may be detected in their feces using a variety of tests. We compared the level of agreement, ease of procedure, and cost among PCR, lateral flow immunoassay, fluorescent antibody, and Kinyoun acid-fast stain direct smear tests. Over the course of 9 mo, 74 calf fecal samples were submitted and tested for C. parvum using all 4 tests. A Fleiss kappa value of 0.813 was obtained, indicating an excellent level of agreement among tests. Overall, the best test based on cost and ease of procedure was the Kinyoun acid-fast stain direct smear.

Microbiomes of Various Maternal Body Systems Are Predictive of Calf Digestive Bacterial Ecology.

Simple SummaryComposition of the bacterial community in a newborn’s gut plays a role in their early development and immune system function. Understanding relationships between the bacterial communities of cows and their offspring can help identify which communities have a greater influence on bacterial community development. We examined bacteria at various sites of the cow at birth and bacteria in their calf’s gut throughout early life to understand their relationship. We found that bacteria in the cow’s reproductive tract, gut, and even milk all served as predictors for calf gut bacteria from birth up to 60 d old. Further exploration of these relationships as well as examining relationships of these bacterial communities with illness could help to prevent disease in calves.Body systems once thought sterile at birth instead have complex and sometimes abundant microbial ecosystems. However, relationships between dam and calf microbial ecosystems are still unclear. The objectives of this study were to (1) characterize the various maternal and calf microbiomes during peri-partum and post-partum periods and (2) examine the influence of the maternal microbiome on calf fecal microbiome composition during the pre-weaning phase. Multiparous Holstein cows were placed in individual, freshly bedded box stalls 14 d before expected calving. Caudal vaginal fluid samples were collected approximately 24 h before calving and dam fecal, oral, colostrum, and placenta samples were collected immediately after calving. Calf fecal samples were collected at birth (meconium) and 24 h, 7 d, 42 d, and 60 d of age. Amplicons covering V4 16S rDNA regions were generated using DNA extracted from all samples and were sequenced using 300 bp paired end Illumina MiSeq sequencing. Spearman rank correlations were performed between genera in maternal and calf fecal microbiomes. Negative binomial regression models were created for genera in calf fecal samples at each time point using genera in maternal microbiomes. We determined that Bacteroidetes dominated the calf fecal microbiome at all time points (relative abundance ≥42.55%) except for 24 h post-calving, whereas Proteobacteria were the dominant phylum (relative abundance = 85.10%). Maternal fecal, oral, placental, vaginal, and colostrum microbiomes were significant predictors of calf fecal microbiome throughout pre-weaning. Results indicate that calf fecal microbiome inoculation and development may be derived from various maternal sources. Maternal microbiomes could be used to predict calf microbiome development, but further research on the environmental and genetic influences is needed.

1416. Assessing the potential for interspecies transmission of Clostridioides difficile on dairy farms

Background Clostridioides difficile (CD) can cause severe colitis in humans and many species of animals. It is thought that farm animals could be a reservoir for CD and that farm workers could therefore be at increased risk of colonization and infection with CD. While pigs and swine farm workers have been shown to be colonized with identical clones of CD, the zoonotic transmission of CD from animals to people has not been definitively demonstrated, and no studies have examined whether dairy farm workers, who are generally in closer contact with animals than swine farmers, are at increased risk of being colonized or infected with CD. The aim of this study was to assess whether dairy calves and farm workers harbored genetically similar isolates of CD.MethodsFirst, we validated a glove-juice protocol to detect CD spores on the hands of farm workers. Volunteers’ hands were inoculated with serially diluted suspensions of non-toxigenic CD organisms, and hand rinsates underwent broth enrichment and anaerobic culture. Second, we collected fecal samples from 5 randomly selected dairy calves (< 7 d of age) from each of 23 farms in southeastern Pennsylvania, northern Maryland, and Delaware. We focused specifically on dairy calves, as the prevalence of CD is highest in this age group. Third, using the glove-juice protocol, we collected hand rinsates from 38 dairy farm workers who work closely with calves. Only 4 of these workers were willing to submit fecal samples along with their hand rinsates. All fecal samples and hand rinsates underwent broth enrichment and anaerobic culture for CD.ResultsValidation of the glove juice protocol showed that CD could be recovered successfully from all hand rinsate dilutions (up to 10-6). When applied to farm workers, this method yielded CD in none of the hand rinsates (0%, 95% CI 0.0-92.2%). CD was also not detected in any of the human fecal samples. However, CD was detected from calf fecal samples on 10 farms (43.5%, 95% CI 20.8%-80.0%).ConclusionWhile the zoonotic transmission of CD cannot be ruled out, our results suggest that contact with dairy animals is not likely to be associated with an increased risk of acquiring CD via the fecal-oral route. The glove-juice protocol appears to be a useful tool for studying the epidemiology of CD in populations where obtaining fecal samples is difficult.Disclosures All Authors: No reported disclosures

Caution at choosing a particular colony-forming unit from faecal Escherichia coli: it may not represent the sample profile.

Data about phylogenetic classification of Escherichia coli colonizing calves, lambs and foals are routinely neglected and restricted to outdated methodologies, even in the context of antimicrobial susceptibility (AS) testing. Thus, the aim of this study was to determine the phylogenetic diversity and the AS profile of E. coli colony-forming units (CFUs) from faecal samples of healthy animals. Five CFUs of E. coli were randomly selected from each faecal culture of calves (n=13), foals (n=13) and lambs (n=13), totalizing 195 CFUs phylo-typed by quadruplex PCR. The AS profile of five CFUs from 15 samples (five from each animal species; n=75 isolates) against nine drugs was determined by agar diffusion test. We found E. coli belonging to all phylo-groups already described, except D group, with the predominance of B1 (65% CFUs; 126/195) in the three-animal species sampled. Most faecal samples of calves (77%; 10/13) and foals (69%; 9/13) harboured both pathogenic and nonpathogenic E. coli. All faecal samples showed CFUs with diverse AS profile, highlighting the ineffectiveness of tetracycline, sulphonamide and ampicillin. As a key point, our data reinforce the importance to select at least four E. coli CFUs for AS testing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides relevant data about the high phylogenetic and antimicrobial susceptibility diversity observed in Escherichia coli colony-forming units (CFUs) from a bacteriological culture of faeces from healthy calves, foals and lambs. The selection pressure exerted by the herd treatment may directly impact the intestinal microflora of animals that have never been treated. Finally, we emphasize the importance of Clinical Laboratory Standards Institute guidelines and we recommended to analyse at least four E. coli CFUs to determine, in particular, the antimicrobial susceptibility profile of faecal isolates, independent of the animal's health status.

Disease burden and associated factors of rotavirus infection in calves in south-eastern part of Bangladesh

Rotaviruses are considered as a major cause in the etiology of diarrhea in young calves worldwide and it causes calf morbidity, mortality and substantial economic losses in the dairy industry. This study reports the determination of the disease burden and first evaluation of associated risk factors of rotavirus infection in calves in Bangladesh. A cross sectional survey was carried out for the approximation of prevalence and associated risk factors of rotavirus infection in neonatal calves in South-eastern part of Bangladesh. From July 2015 to May 2016 a total of 411 calves under the age of 6 weeks from 210 dairy farms were selected using probability sampling scheme. Rotavirus infection was diagnosed from calf fecal samples using a commercial ELISA kit. Different farm and calf level factors were tested by mixed effect multivariable logistic regression models to differentiate factors significantly contributing to the high burden of rotavirus infection in calves in the study area. Overall prevalence of rotavirus in calf feces, irrespective of diarrheic and non-diarrheic, was estimated as 5.11%. Among the hypothesized risk factors, samples from winter season showed higher odds of having the infection compared to summer (OR=6.04). Likewise, higher odds ratios were observed in: &gt;3 weeks of age group compared to ≤ 3 weeks (OR=2.87), taking of first colostrums after 30 minutes to 2 hours of birth in contrast to within 30 minutes (OR=13.92), and finally, calves from 3rd to 4th parity compared to 1st to 2nd parity (OR=6.79). Enteric diseases are a vital health problem in calves that interrupts production benefits with reduced weight gain and increased mortality rates and moreover it has potential to spread. Therefore, it is imperative to determine the disease burden and responsible risk factors to execute effective preventive measures among the population at higher risk.&#x0D; Asian J. Med. Biol. Res. June 2019, 5(2): 107-116

Microbiota of newborn calves and their mothers reveals possible transfer routes for newborn calves' gastrointestinal microbiota.

The intestinal microbiota of newborns plays an important role in the development of immunity and metabolism. In livestock animals, knowledge of the intestinal microbiota is essential not only to prevent diseases but also to optimize weight gain and performance. The aim of our study was to examine faecal samples repeatedly within the first two days of life using 16S rRNA gene High Throughput Sequencing. Additionally, samples from the mouths of the calves and the vaginas, colostrum, and faeces of the dams were included to evaluate possible sources of the calf faecal microbiota. The calf faecal microbiota was highly variable during the first 48 hours post natum (p.n.). Significant changes were found in species diversity and richness, in copy numbers evaluated by qPCR and in predominant bacteria over time. The most pronounced changes occurred between 6 and 24 hours p.n. All calf faecal samples were dominated by Operational Taxonomic Units (OTUs) belonging to the family Enterobacteriaceae. Cow faecal samples showed significantly higher species richness, diversity, number of observed OTUs, and copy numbers compared to all other samples. OTUs belonging to the family Ruminococcaceae were most abundant in cow faecal and vaginal samples. Colostrum was dominated by Enhydrobacter affiliated OTUs. To identify possible inoculation routes for the calf microbiota, we analysed OTU sharing between samples. The calf microbiota during the first two days of life was clearly distinct from the dam’s faecal microbiota. Furthermore, colostrum microbiota clearly differed from calf and cow faecal microbiota and thus most likely does not play an important role as inoculation source for calf microbiota during the first two days of life. In contrast, the cow vaginal and the calf faecal microbiota were more similar, suggesting that some of the calf faecal microbiota may derive from inoculation from the birth canal during birth.

Molecular characterization of Cryptosporidium isolates from diarrheal dairy calves in France.

Cryptosporidium is an obligate intracellular protist parasite infecting a wide range of vertebrate hosts and causes significant intestinal disease in both animals and humans, as some species are zoonotic. Cattle and especially calves have been identified as one of the most common reservoirs of this protist. However, little is known about the genetics of Cryptosporidium in calves in some regions of France. The aim of this study was to detect and isolate Cryptosporidium spp. in faecal samples from naturally infected pre-weaned calves (≤45 days-old) in France. A total of 35 diarrhoeic pre-weaned calf faecal samples were collected from 26 dairy cattle farms in six departments (French administrative provinces). Cryptosporidium presence was established by microscopically screening samples for oocystes with an immunofluorescent (DFA) staining method. DFA-positive samples were then analysed by PCR-RFLP and 18S rRNA gene sequencing to determine species. Cryptosporidium parvum-positive samples were subtyped via nested PCR analysis of a partial fragment of the 60 kDa glycoprotein (gp60) gene product. Data were then integrated into phylogenetic tree analysis. DFA revealed the presence of Cryptosporidium oocysts in 31 out of 35 (88%) samples. Combined with 18S rRNA gene analysis results, C. parvum was detected in 30 samples. Subtyping analysis in 27/30 samples (90%) of the C. parvum isolates revealed two zoonotic subtype families, IIa (24/27) and IId (3/27). Four subtypes were recognised within the subtype family IIa, including the hypertransmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (21/27), IIaA17G3R1 (1/27), IIaA17G1R1 (1/27), and IIaA19G1R1 (1/27). Two subtypes were recognised within the IId subtype family including IIdA22G1 (2/27) and IIdA27G1 (1/27). These findings illustrate the high occurrence of Cryptosporidium in calves in dairy herds and increase the diversity of molecularly characterised C. parvum isolates with the first description of IIaA17G3R1, IIaA19G1R1, and IId subtypes in France. The presence of zoonotic C. parvum subtype families (IIa, IId) in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, and highlights the importance of animal to human cryptosporidiosis transmission risk. Further molecular studies in calves and small ruminants from other French regions are required to better understand the epidemiology of cryptosporidiosis in France.

Evaluation of fecal hormones for noninvasive research on reproduction and stress in humpback whales (Megaptera novaeangliae)

Fecal hormone analysis shows high potential for noninvasive assessment of population-level patterns in stress and reproduction of marine mammals. However, the marine environment presents unique challenges for fecal sample collection. Data are still lacking on collection methodology and assay validations for most species, particularly for those mysticete whales that have variable diets. In this study we tested collection techniques for fecal samples of free-swimming humpback whales (Megaptera novaeangliae), and validated immunoassays for five steroid and thyroid hormones. Resulting data were used for preliminary physiological validations, i.e., comparisons to independently confirmed sex and reproductive state. Pregnant females had significantly higher fecal progestins and glucocorticoids than did other demographic categories of whales. Two possible cases of previously undetected pregnancies were noted. Males had significantly higher fecal testosterone metabolites than nonpregnant females. Fecal glucocorticoids were significantly elevated in pregnant females and mature males compared to nonpregnant females. Calf fecal samples had elevated concentrations of all fecal hormones. Fecal thyroid hormones showed a significant seasonal decline from spring to summer. Though sample sizes were small, and sampling was necessarily opportunistic, these patterns indicate that noninvasive fecal hormone analysis may facilitate studies of reproduction, stress and potentially energetics in humpback whales.