Feces Of Calves Research Articles

Saccharomyces cerevisiae boulardii CNCM I-1079 affects health, growth, and fecal microbiota in milk-fed veal calves

The objective of this study was to investigate the effect of one specific strain of yeast, Saccharomyces cerevisiae boulardii CNCM I-1079 (SCB), on the growth performance, health, and fecal bacterial profile of veal calves. A total of 84 animals were enrolled in an experiment at a commercial veal farm for a total of 7 wk. Calves were fed twice a day with a milk replacer meal during the entire experiment and were randomly assigned to receive daily either SCB supplementation (10 × 109 cfu/d) or a placebo (CON). Individual feed intake and body weight were monitored on a daily and weekly basis, respectively. Fecal samples were collected at arrival to the veal facility (wk 0) and additional samples were taken on d 14 (wk 2) and d 49 (wk 7). These samples were subjected to 16S rRNA gene amplicon sequencing using Illumina MiSeq (Illumina Inc., San Diego, CA) to examine the bacterial profiles and real-time quantitative PCR to quantify Saccharomyces cerevisiae and specific bacterial groups. The significant increase of S. cerevisiae in the feces of SCB calves at wk 2 and 7 compared with wk 0 (respectively 1.7 × 107, 1.2 × 107, and 2.2 × 105 copy number of S. cerevisiae/g of feces) indicates a good survival of that yeast strain along the gastrointestinal tract. Supplementation of SCB did not improve overall growth performance with regard to average daily gain (ADG), final body weight, and feed intake. Nevertheless, a total of 69.1% of nonsupplemented calves had diarrhea and 28.6% experienced severe diarrhea, whereas 50.0% of the calves supplemented with SCB had diarrhea and 9.5% experienced severe diarrhea. With respect to antibiotic use, 89.7% of the diarrheic calves recorded in the CON group were treated, whereas only 66.7% of the SCB diarrheic calves received an antibiotic. In addition, diarrheic calves supplemented with SCB maintained an ADG similar to nondiarrheic animals, whereas the CON diarrheic calves had a significantly lower ADG in comparison with nondiarrheic CON calves. Fecalibacterium was the most predominant bacterial genus in fecal samples of nondiarrheic and diarrheic calves supplemented with SCB, whereas fecal microbiota was predominated by Collinsella in diarrheic calves from the CON group. Live yeast supplementation in milk replacer led to a decrease of diarrhea in milk-fed veal calves and the fecal microbiota of diarrheic calves maintained a healthy community similar to nondiarrheic animals, with Fecalibacterium being the predominant genus.

STRUCTURAL ASSESSMENT OF VIOLATIONS OF THE ADAPTIVE MECHANISMS OF ADRENAL CALVESIN THE ACUTE COURSE OF CRYPTOSPORIDIOSIS

The study was the first to assess the adaptation mechanisms disorders of the adrenals of calves in acute cryptosporidiosis. The cryptosporidiosis in patients with symptoms of diarrhea in the farms of the Republic of Tatarstan was diagnosed through formalin-ether sedimentation and following staining of infectious agents by Ziehl-Neelsen acid fast staining method. For specific identification of cryptosporidium in the feces of calves, an enzyme immunoassay kit, “H&R Crypto Rapid Test”, was used to detect cryptosporidium in feces. The agents of cryptosporidiosis of the genus Cryptosporidium were determined in pathological material taken from sick and dead at the age of 5 to 10 days calves. Histological examination of the organs and tissues of calves with the cryptosporidiosis were carried out using generally accepted method of staining of histological sections with hematoxylin and eosin. Structural studies of adrenal cortex and medulla of calves with the cryptosporidiosis revealed significant changes in synthesized hormonal products profiles. The characteristics of intense glucocorticoid biosynthesis were found in the adrenal cortex at the height of disease, as shown by a sharp expansion in zona fasciculate of the cortex, numerous small and large vacuoles in the cytoplasm of spongiocytes and enhanced profiles of capillary net. Intense excretion of glucocorticoids in hemocirculation maximally activated the catabolic processes in the organisms of sick calves. Small number of adrenocorticocytes, the predominance of small hyperchromic nuclei in them and the absence of characteristics of mitotic activity were not edinzona glomerulosa of the cortex that corresponded to the morphological equivalent of retardation of mineralcorticodes biosynthesis. There had been a dramatic increase in cells with characteristics of apotosis among adrenocorticocytes in all zones of the cortex, including the small in width reticular zone. The small catecholamine-synthesizing cells in the medullary area were represented predominantly by small with sharply clarified cytoplasm and small rounded adrenocytes nuclei and by significantly smaller amount of larger noradrenocytes. The found pathological changes in the cortex and medulla of the adrenals characterized the increase of catabolic processes, vascular, metabolic disorders in organisms of calves with the cryptosporidiosis. In view of disorders of adaptation mechanisms and limited plastic resources, a fast death was observed in newborn calves.

Genetic analysis of bovine viral diarrhea virus in pre-weaned native Korean calves.

Bovine viral diarrhea virus (BVDV), a prominent viral pathogen worldwide, causes substantial economic losses in the cattle industry. BVDV comprises two recognized species, BVDV-1 and BVDV-2, and at least 21 subtypes (1a-1u) for BVDV-1 and four subtypes (2a-2d) for BVDV-2 based on its 5'-untranslated region. This study aimed at investigating the prevalence and genetic analysis of BVDV in calf feces in the Republic of Korea (ROK). We collected fecal samples from 635 pre-weaned native Korean calves aged 1-60days, regardless of diarrhea, and subjected them to RT-PCR and phylogenetic analysis. Thirty-five (5.5%) of the 635 samples were positive for BVDV infection. BVDV was detected in 20, 10, and 5 calves aged 1-20days, 21-40days, and 41-60days, respectively. BVDV was the most frequent in 17 normal feces, followed by 16 diarrheic feces, and 2 hemorrhagic feces. Phylogenetic analysis revealed that 25 samples belonged to BVDV-1b; 1 sample, BVDV-1c; and 9 samples, BVDV-2a. Moreover, the BVDV-1b and BVDV-2a isolates showed genetic variations. BVDV-1b was detected in diarrheic, hemorrhagic, and normal fecal samples. Thus, BVDV-1b is the most prevalent in calves and causes enteric disease with differing severity. BVDV-1c was newly identified in diarrheic calves. Further studies are warranted to elucidate the pathogenesis of BVDV-1c infection and its clinical manifestations. Our results indicate that effective vaccines and control programs against BVDV are required in the ROK.

Cellular immune response and scanning electron microscopy in the evaluation of Moringa leaves aqueous extract effect on Cryptosporidium parvum in buffalo intestinal tissue explants.

Cryptosporidium is an apicomplexan parasite of human and animals and is considered as an important co-factor in neonatal diarrhea. In this study, an explant culture was usedas an in vitro model of buffalo intestine to evaluate the effect of Moringa leaves extract on Cryptosporidium parvum (C. parvum) oocysts using light and scanning electron microscopy and measuring IFN-γ, IL-12 and IL-14 in the culture supernatants. C. parvum oocysts were collected from naturally-infected calf feces, isolated, excysted and then co-inoculated with ileal tissue explants culture medium. The prepared Moringa leaves extract was then introduced to the infected tissues in the concentrations of 100mg/ml and 300mg/ml. After 24h, tissues were collected and processed for light and scanning electron microscopy. Also, culture supernatants were collected for cytokines measurement. C. parvum parasitophorous vacuoles were found attached to the surface of tissue in Cryptosporidium-infected ileal tissue explants. High magnification imaging of ileal tissue explants using scanning electron microscopy showed that Moringa leaves extracts had a great effect on Cryptosporidium-infected ileal tissue explants. There was a high significant (P < 0.001) increase in IFN-γ, IL-12 and IL-14 (375, 275 and 90pg/ml, respectively) in the supernatants of infected non-treated ileal tissue explant culture plate wells compared to the control non-infected ones (74.66, 75 and 50pg/ml, respectively). A concentration of 100mg/ml Moringa extract exhibited the highest anticryptosporidial effect causing a significant decrease in IFN-γ, IL-12 and IL-14 levels (225, 150 and 65pg/ml, respectively) compared with supernatants of infected non-treated ileal explant culture plate wells. In this study, explant culturing of buffalo ileal tissues allowed investigating the pathogenesis of cryptosporidiosis using light and scanning electron microscopy and studying changes in cytokine levels in tissues with and without Moringa leaves extract treatment. This model could helpto understand the regulation of intestinal secretory and inflammatory responses, and could be useful for the screening of potential anticryptosporidial candidate compounds.

Diversity of Plasmids and Genes Encoding Resistance to Extended Spectrum Cephalosporins in Commensal Escherichia coli From Dutch Livestock in 2007-2017.

Extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase (pAmpC) genes confer resistance to extended spectrum cephalosporin’s. The spread of these genes is mostly facilitated by plasmid-mediated horizontal transfer. National surveillance activities to detect ESBL/pAmpC-producers in commensal bacteria from livestock are in place in the Netherlands since several years. This study aimed at reporting gene and plasmid diversity of commensal ESBL/pAmpC-producing Escherichia coli isolated from healthy animals during surveillance activities between 2007 and 2017. A collection of 2304 extended-spectrum cephalosporin-resistant (ESC-R) E. coli isolated from feces of broilers, dairy cattle, slaughter pigs, turkeys, ducks, and veal calves was investigated and ESBL/pAmpC genes were determined. Gene location of a selection of 473 E. coli isolates was determined and typing of plasmids linked to the ESBL/pAmpC genes was performed. Twenty-two different ESBL/pAmpC genes were identified with blaCTX-M-1 being the most prevalent gene in livestock (43.7%), followed by blaCMY -2 and blaSHV -12, independent of the animal source. Prevalence of typically human associated blaCTX-M-15 was highest in cattle. Less than 10% E. coli isolates owed their ESC-R phenotype to promoter mutations of the chromosomal ampC gene. Majority (92%) of ESBL/pAmpC genes analyzed were plasmid located, with IncI1α being the most represented plasmid family in isolates from all animals, followed by IncF (veal calves, dairy cattle and slaughter pigs), IncK (broilers and laying hens), IncX1 in broilers, and emerging IncX3 in broilers and dairy cattle. Prevalence and molecular diversity of ESC-R E. coli isolated from livestock over an 11-year period revealed a composite scenario of gene-plasmid combinations.

Virulence factors and phylotyping of Escherichia coli isolated from non-diarrheic and diarrheic water buffalo calves

ABSTRACT: This study aimed to determine the virulence factors, phylogenetic groups, and the relationships between pathovars and phylogenetic groups of E. coli strains isolated from feces of buffalo calves. A total of 217 E. coli strains were obtained from feces after culture and were screened by PCR for detection of virulence factors EAST-1, enterohemolysin, Saa, CNF2, F41, F5, STa, intimin, Stx1 and Stx2. One hundred and thirty-four isolates were positive for one or more virulence factors: eighty-four from diarrheic animals, and fifty from non-diarrheic calves. The pathovars of E. coli identified in diarrheic feces were ETEC (F5+) (2/84), NTEC (16/84), STEC (20/84), EPEC (3/84), EHEC (3/84), and EAEC (EAST-1+) (33/84). Pathovars identified in non-diarrheic animals were NTEC (21/50), STEC (17/50), EHEC (1/50) and EAEC (7/50). E. coli strains positive for EAST-1 (P=0.008) and phylogroup C (P = 0.05) were associated with the presence of diarrhea. Phylogenetic analysis showed that 58.95% of the isolates belonged to phylogroup B1, followed by E (9.70%), B2 (5.90%), C (5.90%), D (5.22%), A (2.24%), and F (1.50%). Phylogroup B1 predominated in pathogenic E. coli isolated from water buffalo, and phylogroup C constituted an enteropathogenic E. coli for water buffalo calves.

Prevalence of Shiga toxin-producing Escherichia coli in pasture-based dairy herds.

Shiga toxin-producing Escherichia coli strains (STEC) are food-borne pathogens. While E.coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non-O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E.coli O-serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture-based methods. Fresh farm dairy effluent (FDE) (n=36) and composite calf faeces (n=12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx-negative populations present in fresh dairy effluent and calf faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E.coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture-based farms. Such information is required to develop risk-assessment models aiming at limiting transmission of these STEC to human.

Prevalence and Antimicrobial Resistance of Escherichia coli, Enterococcus spp. and the Major Foodborne Pathogens in Calves in Latvia.

The aim of the present study was to detect the prevalence and antimicrobial resistance of fecal indicators and major foodborne pathogens in feces of calves and to identify the factors associated with increased prevalence of resistant bacteria on farms. Altogether, 180 rectal swabs were collected from 18 farms in Latvia. Samples were investigated to detect the prevalence and antimicrobial resistance of Escherichia coli, Enterococcus spp., Listeria monocytogenes, Yersinia enterocolitica, Salmonella spp., Staphylococcus aureus, and Campylobacter spp. Among all, 64% (74/110) of commensal E. coli, 100% (78/78) Enterococcus faecalis and 96% (22/23) Enterococcus faecium isolates were resistant at least to one antibiotic. The prevalence of extended-spectrum β-lactamase (ESBL)/AmpC-producing E. coli were 11.1% (20/180) with blaCTX-M, blaTEM, and blaCMY genes identified. Campylobacter jejuni (12.8%, 23/180) and Campylobacter coli (2.8%, 5/180) were the most resistant to tetracycline (61%, 14/23; 100%, 5/5) and fluoroquinolones (61%, 14/23; 100%, 5/5). Prevalence of L. monocytogenes was 0.6% (1/180) and S. aureus 1.7% (3/180). All samples were Salmonella and Y. enterocolitica negative. Farm size, bought calves, contact with other calves, and antimicrobial treatment of cows were associated with increased prevalence of resistant E. coli and Enterococcus spp. Despite the low overall usage of antimicrobials in Latvia, the high rates of antimicrobial resistance in fecal indicators and Campylobacter, in addition to the high prevalence of ESBL-producing E. coli, highlights the necessity for the prudent use of antimicrobials in dairy farms in Latvia.

Quantification of Clostridioides (Clostridium) difficile in feces of calves of different age and determination of predominant Clostridioides difficile ribotype 033 relatedness and transmission between family dairy farms using multilocus variable-number tandem-repeat analysis

BackgroundCommunity acquired Clostridioides (Clostridium) difficile infection (CA-CDI) is a significant health problem in human and veterinary medicine. Animals are often considered as potential reservoirs for CA-CDI. In Europe, family farming is the most predominant farming operation, with a complex interaction between animals and the community. Therefore, it is pertinent to evaluate transmission patterns of C. difficile on such prominent European farming model.Fecal samples from calves (n = 2442) were collected biweekly over a period of one year on 20 mid-size family dairy farms. Environmental samples (n = 475) were collected in a three month interval. Clostridioides difficile was detected using qPCR in 243 fecal samples (243/2442); positive samples were then quantified. Association between prevalence/load of C. difficile and age of the calves was estimated with logistic regression model. Most common C. difficile isolate from calves (n = 76) and the environment (n = 14) was C. difficile ribotype 033, which was further analyzed using multilocus variable-number tandem-repeat analysis (MLVA) to assess intra- and between-farm relatedness.ResultsClostridioides difficile was detected in feces of calves less than 24 h old. Results showed a non-linear statistically significant decrease in shedding load of C. difficile with age (P < 0.0001). A nonlinear relationship was also established between the number of calves and the farm C. difficile prevalence, whereas the prevalence of C. difficile ribotype 033 increased linearly with the number of calves. MLVA revealed close intra-farm relatedness among C. difficile ribotypes 033. It also revealed that the between-farms close relatedness of C. difficile ribotypes 033 can be a direct result of farm to farm trade of calves.ConclusionsImplementation of better hygiene and management measures on farms may help decrease the risk of spreading CA-CDI between animals and the community. Trading calves older than 3 weeks would decrease the possibility C. difficile dissemination in the community because of lower prevalence and lower load of C. difficile in feces.

Diet Supplementation With Pomegranate Peel Extract Altered Odorants Emission From Fresh and Incubated Calves' Feces

Emissions of odorous volatile organic compounds (VOCs) from livestock manure are of increasing environmental concern. Recent approaches in cattle nutrition and health make use of Mediterranean fruit byproducts, such as concentrated pomegranate peel extract (CPE) that carry antioxidant activities, which in turn may alter manure properties. This study explored the effect of CPE on odorants emission from beef calves feces. Fourteen calves were randomly assigned to control (n=7) and pomegranate (n=7) treatments. The latter was supplemented with 4% CPE in milk until weaning at the age of 60d. Following weaning, 4% CPE was added to calves ration, on dry matter basis. The control treatment received only milk or solid feed, respectively. Fresh feces of the four treatments (control/pomegranate; before/after weaning) were sampled twice, 2-3w before and 4-5w after weaning, and then incubated (28°C) for 0, 7, 14, and 30d. Sub-samples were placed in a flux chamber (37°C) and VOCs collected on thermal desorption (TD) tubes followed by TD-GC-MS analysis. In all treatments, flux quantities followed the general order of volatile fatty acids (VFAs) > alcohols > phenolic & aromatic > sulfuric > esters > aldehydes. Total VOCs, especially VFA fluxes, peaked on day 7 in correspondence with pH dynamics. The fractional contribution of alcohols, phenolic & aromatic and sulfuric VOCs generally increased during incubation. After weaning, short-chain VFAs flux was 5.2 times higher and the pH was 1.26 units lower in the pomegranate treatment (average on days 7&14), suggesting increased fermentation due to possible effect of CPE on gastrointestinal microflora. An automated ribosomal intergenic spacer analysis of fresh and incubated fecal DNA confirmed association between microbial fingerprinting and short chain VFAs, phenolic and sulfuric VOCs. Odorants emission after weaning, expressed as odor activity values, was 2 times higher in the pomegranate treatment (average on days 7&14) and generally dominated by VFAs, while in other treatments the contribution of phenolic and sulfuric odors increased as incubation proceeded. In conclusion, diet supplementation with CPE may be adopted with the purpose of increasing calves health and production, but it may alter odor characteristics of feces and increase feedlot nuisance if not managed properly.

Adherence to and invasion of HeLa cells by Campylobacter spp. strains isolated from animals

ABSTRACT: The objective of this study was to evaluate the adherence to and invasion of HeLa cells by Campylobacter spp. strains (total n=63) isolated from chickens (n=4), dogs (n=4), non-human primates (n=16), pigs (n=9), calf feces (n=18), and bovine genital tracts (n=12). Thirty-two strains adhered to and 13 invaded HeLa cells. Invasive strains included 1 of 4 dog isolates, 4 of 16 non-human primate isolates (2 C. jejuni and 2 C. coli), 1 of 9 C. coli strains isolated from pigs, and 7 of 18 C. fetus subsp. fetus isolated from calf feces. Only 25% of chicken and dog isolates and 23% of pig isolates were able to adhere to HeLa cells, a property of 65% of strains obtained from calf feces and 83% of bovine genital tract-isolated strains. The adherent phenotype was observed in 5 of 19, 6 of 15, and 21 of 29 strains of C. jejuni, C. coli, and C. fetus subsp. fetus, respectively, whereas 3 of 19, 3 of 15, and 7 of 29 strains were additionally able to invade HeLa cells, respectively. C. jejuni, C. coli, and C. fetus subsp. fetus strains isolated from animal feces are able to adhere and invade HeLa cells, whereas C. fetus subsp. fetus strains isolated from the bovine genital tract were not invasive in HeLa cells. The present study showed that C. jejuni isolated from primates and dogs, C. coli isolated from non-human primates and pigs, and C. fetus subsp. fetus isolated from calf feces have the ability to adhere to and to invade HeLa cells. Moreover, the lack of invasive ability by C. fetus subsp. fetus strains isolated from the bovine genital tract could be important in the pathogenesis of the genital tract diseases caused by this bacterium.

Molecular detection of integron and antimicrobial resistance genes in multidrug resistant Salmonella isolated from poultry, calves and human in Beni-Suef governorate, Egypt

Antibiotic resistant Salmonella represent a public health threat worldwide. The current study was delineated to determine Salmonella spp. prevalent in poultry droppings (N: 14 flocks), calves feces (N: 90) and human stool samples (N: 80) from live-stock contacts as well as poultry handler in Beni-Suef governorate, Egypt. Phenotypic and genotypic screening for the presence of antimicrobial resistance was applied. Isolation rates were 57.1%, 5.6% and 2.5% for poultry, calves and humans, respectively. S. Megherafelt. S. Kentucky, S. Amsterdam, S. Nigeria, S. Paratyphi: B2, S. Alamo, S. Madras and S. Haifa were the predominant serovars in poultry flocks under investigation. The predominant serovar from calves’ samples was S. Kentucky while S. Infantis and S. Paratyphi: B2 were detected in human samples. The results of antimicrobial disc diffusion revealed the presence of multiple drug resistance (MDR) in100% of Salmonella isolates. PCR confirmed the existence of class 1 integron and the following gene cassettes in the resistant strains, blaTEM and blaSHV for β-lactams, aadA2 for aminoglycosides (streptomycin), tetA and tetB for tetracycline, sull for Sulfamethoxazole, dfrA for trimethoprim, floR for chloramphenicol and mphA for macrolides.

Probiotic properties of native Lactobacillus spp. strains for dairy calves.

The use of native microorganisms with probiotic capacity is an alternative tool for the treatment and prevention of several diseases that affect animals, such as neonatal calf diarrhoea. The selection of probiotic strains within a collection is based on different in vitro and in vivo assays, which predict their potential. The aim of this study was to characterise a group of native Lactobacillus spp. strains isolated from faeces of healthy calves using an in vitro approach and to assess their ability to colonise the gastrointestinal tract (GIT) of calves. Native Lactobacillus spp. strains were evaluated on their capacity to survive low pH conditions and bile salts presence, biofilm formation and adhesion to both mucus and Caco-2 cells. Based on the in vitro characterisation, four strains (Lactobacillus johnsonii TP1.1, Lactobacillus reuteri TP1.3B, L. johnsonii TP1.6 and Lactobacillus amylovorus TP8.7) were selected to evaluate their capacity to colonise and persist in the GIT of calves. The assessment of enteric persistence involved an in vivo assay with oral administration of probiotics and quantification in faeces of the administered bacterial species with real-time quantitative PCR (qPCR). The study was conducted using 15 calves (1-month-old) which were divided into five groups of three animals, four of which were treated with four different selected strains and one was the control group. Strains TP1.3B and TP1.6 managed to persist in treated animals until ten days after the end of the administration period, indicating that they could be promising candidates for the design of probiotics for calves.

Evaluation of three commercial rapid kits to detect Cryptosporidium parvum in diarrhoeic calf stool

The aim of this study was to evaluate three commercially available rapid immunochromatographic tests for detection of Cryptosporidium parvum antigens in faeces of naturally infected neonatal diarrhoeic calves. FASTest® CRYPTO strip, FASTest® CRYPTO-GIARDIA Strip and TETRASTRIPS® were compared for their sensitivity, specificity, positive predictive value and negative predictive value using a cumulative positivity as gold standard. In addition, the agreement between each test and the gold standard was evaluated by Cohen’s Kappa (k) value. The highest infection rate was observed by FASTest® CRYPTO-GIARDIA Strip (65.15%), followed by FASTest® CRYPTO strip (63.64%) and TETRASTRIPS® (56.06%,). A very good diagnostic performance of all the three tests was observed. FASTest® CRYPTO strip (k = 0.935) and FASTest® CRYPTO-GIARDIA Strip (k = 0.968) had the highest sensitivity (100%) while TETRASTRIPS® (k = 0.875) had the highest specificity (100%). Eimeria spp oocysts were present in six samples but cross-reaction with this protozoan was not observed. These assays were not time-consuming and very easy to perform and to read. Based on our results, we recommend the use of FASTest® CRYPTO strip, FASTest® CRYPTO-GIARDIA Strip or/and TETRASTRIPS® for detection of C. parvum antigens in faeces of neonatal diarrhoeic calves.

Production of chitinase from Escherichia fergusonii, chitosanase from Chryseobacterium indologenes, Comamonas koreensis and its application in N-acetylglucosamine production

The important platform polysaccharide N-acetylglucosamine (GlcNAc) has great potential to be used in the fields of food, cosmetics, agricultural, pharmaceutical, medicine and biotechnology. This GlcNAc is being produced by traditional methods of environment-unfriendly chemical digestion with strong acids. Therefore, researchers have been paying more attention to enzymatic hydrolysis process for the production of GlcNAc. Hence, in this study, we isolated novel chitinase (Escherichia fergusonii) and chitosanase (Chryseobacterium indologenes, Comamonas koreensis) producing strains from Korean native calves feces, and developed the potential of an eco-friendly microbial progression for GlcNAc production from swollen chitin and chitosan by enzymatic degradation. Maximum chitinase (7.24±0.07U/ml) and chitosanase (8.42±0.09, 8.51±0.25U/ml) enzyme activity were reached in submerged fermentation at an optimal pH of 7.0 and 30°C. In this study, sucrose, yeast extract, (NH4)2SO4, and NaCl were found to be the potential enhancers of exo-chitinase activity and glucose, corn flour, yeast extract, soybean flour, (NH4)2SO4, NH4Cl and K2HPO4 were found to be the potential activator for exo-chitosanase activity. Optimum concentrations of the carbon sources for enhanced chitinase activity were 9.91, 3.21, 9.86, 1.66U/ml and chitosanase activity were 1.63, 1.13, 2.28, 3.71, 9.02, 4.93, and 2.14U/ml. These enzymes efficiently hydrolyzed swollen chitin and chitosan to N-acetylglucosamine were characterized by thin layer chromatography and were further confirmed by high-pressure liquid chromatography. From a commercial perspective, we isolated, optimized and characterized exochitinase from Escherichia fergusonii (HANDI 110) and chitosanase from Chryseobacterium indologenes (HANYOO), and Comamonas koreensis (HANWOO) for the large-scale production of GlcNAc facilitating its potential use in industrial applications.

Molecular epidemiology of coronavirus in faeces of Brazilian calves and Peruvian camelid herds.

The enteric disorders represent a serious hazard for bovine and camelid breeding. The aim of this study was to examine the frequency of detection and molecular characteristics of enteric coronavirus (CoV) infections in cattle, alpaca, and llama herds bred in family-based farms in Brazil and Peru. Stool samples were collected from calves from Brazil and camelids from Peru for detection and characterization of CoV by reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis. 46.5% (47/101) samples from calves and 26.8% (70/261) from alpaca tested positive for CoV. All strains belong to lineage A1 of the Betacoronavirus genus. Phylogenetic analysis showed high identity between CoV strains detected in calves and alpacas. This study characterised CoV strains from dairy cattle herds in the state of Rio de Janeiro, Brazil, and indicated that this virus is spread among the state herds. The results also indicate widespread circulation of CoV among the alpacas of Cuzco, Peru.

Identification of Virulence Factors of Escherichia coli Isolates from Fecal Samples of Calves in Southern Brazil

Background: Escherichia coli (E. coli) is an enteropathogen that commonly causes diarrhea in calves. However, not all E. coli isolates are pathogenic. The aim of this study was to identify E. coli virulence factors derived from fecal samples collected in the southern region of Rio Grande do Sul state (RS) from calves with and without diarrhea, as well as investigate the antimicrobial susceptibility of E. coli isolates from calves with diarrhea.Materials, Methods &amp; Results: Forty stool samples were collected in 12 farms, each one from calves having one day to six months of age, with and without diarrhea. The total DNA of from these isolates was extracted and a PCR using primers specific for the virulence factors Stx1, Eae, F41, F5 and STa was conducted. The susceptibility testing used the disk diffusion method and the susceptibility profile was evaluated against the following antimicrobials: ampicillin, penicillin, chloramphenicol, enrofloxacin, gentamicin, trimethoprim, sulfonamide, tetracycline and streptomycin. From all calves, 15 (15/40, 37.5%) had diarrheal stools and 25 (25/40, 62.5%) had normal or semi-liquid stools. Twelve (12/40; 30%) E. coli isolates showed at least one virulence factor. These factors were found in four isolates (4/15; 26.6%) from diarrheal stools and eight isolates (8/25; 28.5%) from normal stool. The Stx1 factor was identified in five isolates (5/40; 12.5%), and the Eae and the Sta factors in one (1/40; 0.2%) and in atypical associations between Stx1 and Eae and also between Eae and F41 in two isolates (2/40; 0.5%). Also, the Eae and Sta factors were identified in one isolate (1/40; 0.2%). The susceptibility test showed resistance to penicillin and tetracycline in 93% and 80% of the tested isolates, respectively.Discussion: The identification of virulence factors is necessary because E. coli is an enterobacterium present in calves gastrointestinal tract, to prove its pathogenicity. The virulence factor most commonly found in E. coli isolates derived from feces of calves with and without diarrhea in the southern region of the RS was the Stx1 (Shiga toxin-producing E. coli STEC). It is likely that the highest occurrence of E. coli isolates positive for the Stx1 virulence factor was due to the fact that cattle were the main reservoirs of this type of bacteria. The occurrence of enterohemorrhagic E. coli (EHEC) in animals of nine and 34 days of life, respectively, is highlighted. Studies have shown that contamination of animal foods with EHEC can cause enteric disorders, hemorrhagic colitis, and uremic hemolytic syndrome (UHS) in humans. Although in the present study the identification of the Stx2 factor was not performed, authors describe that the presence of the genes encoding Stx2 and Eae is determinant in the occurrence of UHS. In the susceptibility test, it was observed that E. coli isolates from diarrheal stools showed resistance to antimicrobials penicillin (10 mg) and tetracycline (30 mg) [93% and 80%, respectively], ampicillin (10 mg) [47%], streptomycin (10 mg) [47%], trimethoprim (5 mg) [47%] and sulfonamide (300 mg) [53%]. Although the percentage of antimicrobial resistance varies among studies, it is believed that the indiscriminate use of antimicrobial therapies as a common practice among rural properties contributes to bacterial resistance to these drugs. The sensitivity profile to antimicrobials showed that the analyzed Escherichia coli isolates are resistant to the antimicrobials commonly used for diarrhea treatment in the southern region of the Rio Grande do Sul state, Southern Brazil.

Impact of Saccharomyces cerevisiae boulardii CNCMI-1079 and Lactobacillus acidophilus BT1386 on total lactobacilli population in the gastrointestinal tract and colon histomorphology of Holstein dairy calves

There is interest in the use of direct-fed microbials (DFM) as substitutes for antibiotic growth promoters in farm animal production. However, little information exists on their effects in dairy calves. The aims of this study were to: 1) determine the viability of supplemental DFM in feed and throughout the gastrointestinal tract (GIT) of calves; 2) evaluate the effect of DFM on total lactobacilli population in the digestive tract and in feces of calves; and 3) examine the influence of DFM on colon histomorphology and overall calf performance. Forty eight calves (2–7days old) were randomly allocated to four treatments as follows: 1) Control (CTRL) fed milk replacer (MR) followed by starter feed (SF); 2) CTRL supplemented with Saccharomyces cerevisiae boulardii CNCMI-1079 (SCB; 7.5×108 colony forming units (CFU)/L MR+3×109 CFU/kg SF); 3) CTRL supplemented with Lactobacillus acidophilus BT1386 (LA; 2.5×108 CFU/L MR+1×109 CFU/kg SF); and 4) CTRL supplemented with antibiotics (ATB) chlortetracycline and neomycin (528 and 357mg/L MR, respectively), and chlortetracycline (55mg/kg SF). Four calves per treatment were euthanized on day 33 (pre-weaning) and an additional four calves per treatment on day 96 (post-weaning) to sample different sections of the GIT. The effects of treatments were analyzed following a completely randomized design with repeated measures and Tukey adjustments for multiple comparisons. The DFM (SCB and LA) remained viable in the commercial product throughout the period of the experiment. Viable SCB was recovered throughout the GIT and in the feces pre- and post-weaning. There was no effect of DFM on total lactobacilli population in the GIT of calves. However, feces of SCB-treated calves showed a greater lactobacilli population compared with CTRL (P<0.01) around weaning. In the pre-weaning period, the distribution of lactobacilli population differed along the digestive tract (colon > ileum content > rumen > ileum mucosa; P<0.001). The lactobacilli population was significantly reduced in all compartments (P=0.02) post-weaning compared to pre-weaning, except in the rumen. Crypts depth and width of the colon decreased (P<0.01) whereas number of goblet cells containing neutral mucins tended to increase (P=0.058) while acidic mucins increased (P<0.05) in SCB- and ATB-treated calves compared with CTRL and LA-treated calves. Overall, growth performances were not affected by treatment. Feeding SCB stimulated total lactobacilli population around weaning and altered colon morphology with potential beneficial effects during the early period of growth in calves. Our findings suggest that SCB would deserve more attention as a modulator of the gastrointestinal health in young dairy calves.

Detection of Bovine Coronavirus in Healthy and Diarrheic Dairy Calves.

Background BCoV is identified in both healthy and diarrheic calves, complicating its assessment as a primary pathogen.ObjectivesTo investigate the detection rates of bovine coronavirus (BCoV) in feces of healthy and diarrheic calves and to describe the usefulness of a pancoronavirus reverse transcriptase (RT) PCR (PanCoV‐RT‐PCR) assay to identify BCoV in samples of diarrheic calves.AnimalsTwo hundred and eighty‐six calves <21 days. Calves with liquid or semiliquid feces, temperature >39.5°C, and inappetence were considered as cases, and those that had pasty or firm feces and normal physical examination were designated as controls.MethodsProspective case–control study. A specific BCoV‐RT‐PCR assay was used to detect BCoV in fecal samples. Association between BCoV and health status was evaluated by exact and random effect logistic regression. Fecal (n = 28) and nasal (n = 8) samples from diarrheic calves were tested for the presence of BCoV by both the PanCoV‐RT‐PCR and a specific BCoV‐RT‐PCR assays. A Kappa coefficient test was used to assess the level of agreement of both assays.Results BCoV was detected in 55% (157/286) of calves; 46% (66/143), and 64% (91/143) of healthy and diarrheic calves, respectively. Diarrheic calves had higher odds of BCoV presence than healthy calves (OR: 2.16, 95% CI: 1.26 to 3.83, P = 0.004). A good agreement between PanCoV‐RT‐PCR and BCoV‐RT‐PCR to detect BCoV was identified (κ = 0.68, 95% CI: 0.392 to 0.967; P < 0.001).Conclusions and Clinical Importance BCoV was more likely to be detected in diarrheic than healthy calves. The PanCoV‐RT‐PCR assay can be a useful tool to detect CoV samples from diarrheic calves.

Composition of the microbiota in forestomach fluids and feces of Japanese Black calves with white scours1

The objective of this study was to characterize the composition of the forestomach and fecal microbiota in Japanese Black calves with white scours. Forestomach fluid, feces, and peripheral blood were collected from healthy calves (n = 5; age 10 ± 2 d) and scouring calves (n = 5; age 10 ± 1 d) on the day on which white scours occurred. The pH and concentrations of VFA, lactic acid, and ammonia nitrogen (NH3–N) of the forestomach fluids were determined. Microbiota composition and gene copy numbers in the forestomach fluid and feces were analyzed by 454 pyrosequencing and quantitative real-time PCR (qPCR), respectively. The cytokine mRNA level in peripheral leukocytes was evaluated by qPCR. The pH of the forestomach fluid of the scouring calves tended to be higher than that of the healthy calves (P = 0.056). No significant difference was detected in the total VFA, lactic acid, or NH3–N concentrations in the forestomach fluids of the 2 groups. Firmicutes, Bacteroidetes, and Proteobacteria were the predominant phyla in the forestomach fluid and feces. At the genus level, the relative abundance of Oscillibacter in the forestomach fluid was significantly higher in the scouring calves (P < 0.05) and the relative abundance of Faecalibacterium in the feces was significantly higher than that in the forestomach in the healthy calves (P < 0.05). Furthermore, the bacterial diversity indices of feces were lower in the scouring calves. Quantitative PCR amplification using some of the primer pairs failed in the forestomach fluid and feces in both groups. These results suggested that fermentation in the forestomach may affect the occurrence of white scours, resulting in changes in the composition and diversity of the forestomach fluid and fecal microbiota in Japanese Black calves.