Bovine Diarrhea Research Articles

Characterisation of Early Positive mcr-1 Resistance Gene and Plasmidome in Escherichia coli Pathogenic Strains Associated with Variable Phylogroups under Colistin Selection.

An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes; however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.

Severe outbreak of bovine neonatal diarrhea in a dairy calf rearing unit with multifactorial etiology.

This study describes the etiological diversity observed in a severe neonatal diarrhea outbreak with morbidity and mortality rates of 80 and 20%, respectively, with detection of mixed infections with viral, bacterial, and protozoan disease agents in a dairy calf rearing unit. Diarrheic fecal samples were collected from eight 5 to 18 days of age calves and were submitted to the investigation of the presence of rotavirus A (RVA), bovine coronavirus (BCoV), bovine kobuvirus (BKV), bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), enteropathogenic Escherichia coli (ETEC), Salmonella sp., and Cryptosporidium spp. Fragments of the small intestine of one calf with diarrhea that spontaneously died were submitted for histopathological analyses. The most frequent infectious agent detected in diarrheic fecal samples was BKV (8/8—100%), followed by RVA (5/8—62.5%), BVDV (5/8—62.5%), Cryptosporidium parvum (5/8—62.5%), ETEC (4/8—50%), and Cryptosporidium ryanae (1/8—12.5%). These etiological agents were found in mixed infections with two or more pathogens per diarrheic fecal sample. The association of viral and protozoan pathogens was the most frequently identified (37.5%) in these samples, followed by viral and bacterial (25%); viral, bacterial, and protozoan (25%); and only viral agents (12.5%). BCoV and Salmonella sp. were not identified in the diarrheic fecal samples analyzed. Additionally, histopathology of the small intestine diagnosed chronic lymphocytic enteritis. In conclusion, in calf rearing units, the adoption and strict monitoring of health management practices are critical to the success of this calf creation system.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42770-021-00565-5.

Pre-weaned dairy calf management practices, morbidity and mortality of bovine respiratory disease and diarrhea in China

Controlling disease, maintaining animal well-being, and sustaining production efficiency in Chinese dairy industry will depend on whether enough of China's dairy farmers can adopt proper calf management practices. Consequently, to provide insights into a number of beneficial management practices used by many Chinese dairy farmers, ninety-eight dairy farms, located in the country's three typical climate zones (severe cold zone, SCZ; cold zone, CZ; and hot-summer/cold-winter zone, HSCWZ) were surveyed. The various management practices employed and the morbidity and mortality rates caused by bovine respiratory disease and diarrhea were recorded at each investigated farm. About three quarters of the calves on these farms were housed indoors (either individually or in group pens). The proportion of the calves reared in separate pens (individual indoor or outdoor hutches) was highest (56.20%) at large-scale farms (> 500 cows), followed by 33.3% at medium-scale farms (50–500 cows). A majority of the investigated farms (≥ 68.04%) did not have a system for monitoring either temperature, relative humidity, or air quality. A medium depth (score 2) of bedding was most common at 42.11% of the farms. 87.76% of the farms relied on natural ventilation. 36.10% of the farms did not provide supplemental heating in the calf barn, and the heating methods most often used differed among the three climate zones. Manual milk feeders involving bottles or buckets were more common (83.67%) than automated milk feeders. The mean morbidity and mortality rates of pre-weaned calves infected with bovine respiratory disease were 11.73% (95% CI:10.2–13.23%) and 3.65% (95% CI: 2.85–4.45%), while those infected with diarrhea were 14.17% (95% CI: 12.46–15.88%) and 3.61% (95% CI: 2.97–4.25%). The rates were highest in the SCZ, in spring and winter, and among group-housed calves. These results should provide insights that could lead to better management practices improvements that will help to sustain not only China's dairy farms but also dairy farms operated in the similar climate zones around the world.

Characterization of Multidrug-Resistant Biofilm-Producing Escherichia coli and Klebsiella pneumoniae in Healthy Cattle and Cattle with Diarrhea

This study describes comparative occurrence and characterization of multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae (KP) in healthy cattle (HC) and cattle with diarrhea (DC) in India. During 2018-2020, 72 MDR isolates, including 35 E. coli (DC: 27; HC 8) and 37 K. pneumoniae (DC: 34; HC: 3), from 251 rectal swabs (DC: 219; HC: 32) were investigated for extended-spectrum beta-lactamase (ESBL), AmpC type β-lactamase and carbapenemase production, antimicrobial susceptibility profile, biofilm production, and efflux pump activity. Fifty-five MDR isolates were ESBL producers (ESBLPs) (DC: 50; HC: 5) and ESBLPs from DC were coresistant to multiple antibiotics. The blaCTX-M gene (50) was the most frequently detected β-lactamases followed by blaAmpC (22), blaTEM1 (13), blaCMY-6 (6), blaOXA1 (5), blaPER (2), blaDHA, and blaFOX and blaSHV12 (1 each). Plasmid-mediated quinolone resistance determinants qnrB, qnrS, qnrA, and qepA were detected in 18, 16, 2, and 3 isolates, respectively. Twenty three isolates revealed mutation in gyrA and parC genes. Tetracycline-resistance markers tetA, tetB, tetC, and tetE were detected in 33, 10, 3, and 2 isolates, respectively. Only one of the 41 imipenem-resistant isolates harbored blaNDM-5 and two were colistin-resistant. Altogether, 20 MDR isolates were strong biofilm producers and 19 harbored different virulence factors. This is the first ever report from India on the presence of MDR Enterobacteriaceae with resistance to even last-resort antimicrobials in the bovine diarrhea.

Use of sulfonamides for the treatment of bovine neonatal diarrhea: clinical and performance parameters

This study was developed to evaluate the clinical, hematological, and performance parameters of calves affected by diarrhea caused by the bacterial enteric pathogen Escherichia coli, treated with three different protocols containing sulfonamides. Fourteen Holstein calves were monitored from birth to 60 days of age, and divided into 3 groups. Group 1 (n=5) animals were treated with 80 g of Kaopek®, Ibasa, Brazil, administered orally (BM), dissolved in 160 ml of water every 24 h; Group 2 (n=4) received IM sulfadiazine and trimethoprim (Ibatrim®, Ibasa, Brazil), and Group 3 (n=5) animals were treated with 80 g of Kaopek®, Ibasa, Brazil, which is made up of 16 g of phthalylsulfathiazole, associated with 2.28 g neomycin sulfate, 1.6 g of pectin, and 80 g of kaolin, dissolved in 160 ml of water and administered every 24 h by mouth, in addition to sulfadiazine and trimethoprim (Ibatrim®, Ibasa, Brazil), at a daily dose of 16 mg/kg live weight, through IM injection. During the study, the animals were clinically evaluated, and once they were diagnosed with diarrhea, feces samples were collected to identify the bacterial enteric pathogen, antibiogram, polymerase chain reaction (PCR), and coproparasitological exams. Blood samples were collected to evaluate the hematological profile, and the performance profile was monitored weekly. In the clinical examination, all calves presented a reduction in body temperature (<39.2°C) and some improvement in hydration after treatment (p=0,31). However, group 2 had a better concentration of lymphocytes and TP concerning the other animals, as well as better performance. Besides, E.coli was detected in 100% of feces samples. Thus, the therapeutic protocols with sulfonamides used to treat bovine neonatal diarrhea were effective in the clinical improvement of the animals. Nonetheless, the protocol with systemic therapy using sulfadiazine and trimethoprim (Ibatrim®, Ibasa, Brazil) intramuscularly, provided better performance, with better weight gain, and body development of the animals.

Bacterial Ribonuclease (Binase) Promotes Decontamination of MDBK Cell Cultures From Bovine Diarrhea Virus

Bacterial Ribonuclease (Binase) Promotes Decontamination of MDBK Cell Cultures From Bovine Diarrhea Virus

Colostral immunity as an analytical factor in predicting the development of acute respiratory viral infections in calves

To reduce the incidence of acute respiratory viral infections in cattle, routine vaccination of mother cows is carried out. There is a direct dependence of the passive immunity level in calves on the vaccination efficacy in cows. The paper presents the results of a study of colostral immunity in calves and post-vaccination immunity in cows against the agents of acute respiratory viral infections in agricultural facilities located on the territory of the Ural and Volga Federal Districts. In the farms under study (n = 10), cattle are vaccinated with inactivated vaccines: “COMBOVAC” and “COMBOVAC-R” (OOO Vetbiokhim, Russia), “HIPRABOVIS® 4” (Laboratorios Hipra, S. A., Spain). The study of postvaccinal immunity level in cows showed that the levels of antibodies to infectious bovine rhinotracheitis virus (5.3–8.0 log2), bovine viral diarrhea virus (3.5–4.8 log2), bovine parainfluenza-3 virus (6.8–8.5 log2) and bovine respiratory syncytial virus (4.2-4.5 log2) in cattle confer protection. When evaluating the results of serological diagnostics of passive immunity in calves to acute respiratory viral infections, it was found that the level of colostral antibodies in them is lower than the level of post-vaccination antibodies in cows: to infectious bovine rhinotracheitis virus by 34.2–58.8%; to bovine diarrhea virus by 37.5–45.0%; to bovine parainfluenza-3 virus by 14.7–35.4 and to bovine respiratory syncytial virus by 23.5-42.2%. To ensure epizootic favourable situation, it is proposed to adjust the schedules of vaccination against bovine diseases in herds, infected by acute respiratory viral infections for dairy farms under study.

Nutraceutical intervention with colostrum replacer: Can we reduce disease hazard, ameliorate disease severity, and improve performance in preweaned dairy calves?

The objective of this randomized clinical trial was to determine if an intervention with colostrum replacer (CR; Premolac Plus, Zinpro; 125 g/d fed for 3 d) or a placebo milk replacer (Cows Match, Land O'Lakes Inc.; 125 g/d for 3 d) following a triggered alarm could ameliorate disease bouts in dairy calves. The alarm was set to detect negative deviations of milk intake (20% reduction) or drinking speed (30% reduction) in relation to a calf's 12-d rolling average feeding behavior. Calves were enrolled on this study (n = 42 CR, n = 42 placebo) when they triggered an alarm from d 14 to 50 on the feeder before weaning. Once calves were enrolled, calves received a bottle of either 125 g of CR or milk replacer mixed with 1 L of water for 3 d. Calves were enrolled on the automated milk feeder at age 4.0 ± 2.0 d (mean ± SD), were offered 10 L/d of the same milk replacer fed as a placebo, and had ad libitum access to calf starter measured by automated feeders. Weaning began at d 50, with 50% reduction of milk replacer across 14 d, and an additional 20% reduction for another 7 d before weaning at d 70. Calves were health scored from birth to 2 wk postweaning (88 ± 2.0 d of age) daily for bovine respiratory disease (BRD) and diarrhea, and were weighed and scored using lung ultrasonography twice weekly. A BRD score ≥5 and an area of consolidated lung ≥3.0 cm2 was considered BRD positive; diarrhea was defined as a watery fecal consistency that sifted through the bedding. The effect of CR on BRD and diarrhea likelihood were calculated with logistic models; the 7 d before and after an alarm were fixed effects, with milk intake as a covariate. A Cox proportional hazards model evaluated the effect of CR on the hazard of being positive for BRD and diarrhea following enrollment, with sex as a fixed effect in the diarrhea model. The effect of CR on average daily gain the week following intervention was evaluated with linear modeling with repeated measures, and birth date was a random effect. After intervention, placebo calves had 1.64 (95% CI: 1.11-2.43) times greater odds of having a BRD bout compared with CR calves for the 7 d following intervention. Moreover, placebo calves had 1.50 (95% CI: 1.11-2.08) times greater odds of having lobar lung consolidation in the 7 d after intervention than CR calves. No difference was found in the likelihood of diarrhea in the 7 d after intervention (odds ratio: 0.91; 95% CI: 0.71-1.16). However, placebo calves had a 2.38 (95% CI: 1.30-4.33) times greater hazard of incurring BRD in the 14 d after intervention. Average daily gain was not associated with an intervention with CR for the 7 d after intervention (placebo: 0.73 ± 0.07 kg/d; CR: 0.70 ± 0.08 kg/d; LSM ± SEM). These results suggest CR may reduce BRD likelihood but did not influence growth in calves offered high allowances of milk. Future research should examine which properties of CR ameliorate BRD in calves.

Isolation and Atibiotyping of Pseudomonas spp. Associated with Bovine Diarrhea

Bovine diarrhoea is a multifactorial disease involving infectious and non-infectious factors. Various pathogens of bacterial origin are responsible for the diarrhoea in cattle. Gram-negative bacteria are considered as the most common etiological agents isolated from clinical cases of diarrhea. Among Gram negative bacteria, Pseudomonas is considered as the opportunistic pathogen and associated with various problems of medical and veterinary importance including ear and urinary tract infections in dogs, mastitis and metritis in dairy cows and endometritis in horses. During the present study, 47 faecal samples were collected from the clinical cases of bovine diarrhea in Meerut city, Uttar Pradesh for isolation, identification and antibiotyping of Pseudomonas spp. Out of these faecal samples, Pseudomonas spp. could be isolated from six samples indicating the positivity of 12.77%. All the isolates of Pseudomonas spp. were subjected to in vitro antibiogram testing using 7 antimicrobials viz., enrofloxacin, gentamicin, streptomycin, amikacin, oxytetracyclin, ceftriaxone and metronidazole. All the Pseudomonas isolates were resistant to streptomycin, oxytetracyclin and metronidazole. The antibiotic susceptibility profile of the isolates was for ceftriaxone and gentamicin (50% each) and for enrofloxacin and amikacin (33.33% each). Antimicrobial sensitivity analysis of all the six Pseudomonas isolates revealed 04 different biotypes. The study revealed that isolates were found resistant to a number of antibiotics/antimicrobials ranging from 02 to 07. The isolates showing more susceptibility to enrofloxacin and amikacin may be used for the treatment of bovine diarrhea associated with Pseudomonas spp. This further recommended the use of routine antibiotic sensitivity testing before the treatment to avoid dissemination of antimicrobial resistance among microbes.

Alteration of the gut microbiota in post-weaned calves following recovery from bovine coronavirus-mediated diarrhea.

Bovine coronavirus (BCoV) is associated with severe diarrhea, dehydration, and depression, which result in significant economic damages in the dairy and beef cattle industries worldwide. However, differences in the gut microbiota structure and their correlations with differing physiological parameters between BCoV-infected calves with diarrhea and recovered calves are not well understood. In this study, fecal specimens were collected from 10 post-weaned calves, before and after 2 months of fluid therapy, and the samples were used for microbiota analysis. Following recovery, the alpha-diversity profiles (observed operational taxonomic units [OTUs], and Chao1, Shannon, and Simpson indices) changed significantly when compared with those of calves with diarrhea. Beta-diversity analysis exhibited significant differences in gut microbiota compositions between calves with diarrhea and those in the recovered state. The abundances of eight phyla and thirteen genera in feces changed markedly after restoration of BCoV diarrhea. In addition, our correlation study clearly revealed that increased abundances of the genera Caproiciproducens, Pseudoflavonifractor, and Oscillibacter negatively correlated with serum glucose, and phosphorus levels, but positively correlated with serum chloride in calves with diarrhea, whereas increased abundances of the genera Peptostreptococcaceae;Clostridium (Clostridium cluster XI), Intestinibacter, Cellulosilyticum, Ruminococcus, Romboutsia, Paeniclostridium, Clostridiaceae;Clostridium and Turicibacter in recovered calves showed the opposite pattern. These results suggest that structural changes of the gut microbiota after recovery from BCoV infection correlate with changes in physiological parameters. In conclusion, our data provide evidence of gut microbiota-composition changes and their correlations with the physical profiles of post-weaned calves, before and after fluid therapy for BCoV-related diarrhea.

Prevalence of bovine diarrhea disease from Korean native calves in Jeonbuk western area

Prevalence of bovine diarrhea disease from Korean native calves in Jeonbuk western area

A new passive immune strategy based on IgY antibodies as a key element to control neonatal calf diarrhea in dairy farms

BackgroundNeonatal diarrhea remains one of the main causes of morbi-mortality in dairy calves under artificial rearing. It is often caused by infectious agents of viral, bacterial, or parasitic origin. Cows vaccination and colostrum intake by calves during the first 6 h of life are critical strategies to prevent severe diarrhea but these are still insufficient. Here we report the field evaluation of a product based on IgY antibodies against group A rotavirus (RVA), coronavirus (CoV), enterotoxigenic Escherichia coli, and Salmonella sp. This product, named IgY DNT, has been designed as a complementary passive immunization strategy to prevent neonatal calf diarrhea. The quality of the product depends on the titers of specific IgY antibodies to each antigen evaluated by ELISA. In the case of the viral antigens, ELISA antibody (Ab) titers are correlated with protection against infection in calves experimentally challenged with RVA and CoV (Bok M, et al., Passive immunity to control bovine coronavirus diarrhea in a dairy herd in Argentina, 2017), (Vega C, et al., Vet Immunol Immunopathol, 142:156–69, 2011), (Vega C, et al., Res Vet Sci, 103:1–10, 2015). To evaluate the efficiency in dairy farms, thirty newborn Holstein calves were randomly assigned to IgY DNT or control groups and treatment initiated after colostrum intake and gut closure. Calves in the IgY DNT group received 20 g of the oral passive treatment in 2 L of milk twice a day during the first 2 weeks of life. Animals were followed until 3 weeks of age and diarrhea due to natural exposure to infectious agents was recorded during all the experimental time.ResultsResults demonstrate that the oral administration of IgY DNT during the first 2 weeks of life to newborn calves caused a delay in diarrhea onset and significantly reduced its severity and duration compared with untreated calves. Animals treated with IgY DNT showed a trend towards a delay in RVA infection with significantly shorter duration and virus shedding compared to control calves.ConclusionsThis indicates that IgY DNT is an effective product to complement current preventive strategies against neonatal calf diarrhea in dairy farms. Furthermore, to our knowledge, this is the only biological product available for the prevention of virus-associated neonatal calf diarrhea.

The Effect of Co-Administration of Inactivated Vaccines Against Bovine Respiratory Disease and Neonatal Calf Diarrhoea

Abstract A pilot study was performed to evaluate the safety and serological responses after co-administration of two multivalent inactivated vaccines to pregnant cattle. One vaccine was directed against bovine respiratory disease (BRD) and contained antigens of bovine respiratory syncytial virus (BRSV), parainfluenza 3 virus (PI3) and Mannheimia haemolytica (Mh). The second vaccine targeted neonatal calf diarrhoea (NCD) and was composed of inactivated antigens of bovine rotavirus (BRV), bovine coronavirus (BCV) and E. coli. The use of these combinations have been used more and more by veterinary practitioners as there exist some clear evidence that both vaccines improves the passive protection via the colostrum for the relevant pathogens. However, up until now, no safety or efficacy data has been available concerning such co-administrations. The safety of both vaccines and the serological responses to the BRD vaccine has been evaluated when used at the same time, but without mixing and compared to the responses to the administration of each vaccine independently. There was no evidence of any negative effect on calving or calf health in any of the vaccinated animals. The antibody levels against BRSV and Mh in the sera of the calves from cows vaccinated with both vaccines were not significantly different from the levels in the sera of calves vaccinated with the BRD vaccine alone. The results from this pilot study demonstrated that the co-administration of the two multivalent inactivated vaccines had no detrimental effect on the safety or serological responses to the BRD vaccine compared to the independent use of the vaccines.

Health and growth of veal calves provided a fatty acid supplement and a dry teat

Veal calves are at a high risk of disease early in life, which can lead to poor growth. Research is needed to determine interventions that can reduce disease and promote the growth of veal calves. The aim of this study was to determine the effects of fatty acid supplementation and the provision of a dry teat on the incidence of bovine respiratory disease (BRD), navel inflammation, and diarrhea, as well as calf growth. Upon arrival to a commercial veal facility (d 0), 240 Holstein bull calves from 2 cohorts were randomly assigned to 4 treatments using a 2 × 2 factorial arrangement (n = 60/treatment): (1) milk replacer (MR) without fatty acid supplementation and without access to a dry teat (control), (2) MR with fatty acid supplementation (NeoTec5g, Provimi, Brookville, OH) and without access to a dry teat (FAS), (3) MR without fatty acid supplementation and with access to a dry teat (TT), or (4) MR with fatty acid supplementation and with access to a dry teat (FAS+TT). Calves were housed in individual pens from 0 to 9 wk and then paired by treatment at wk 9. Milk replacer was fed twice daily using a step-up program. Fatty acid supplement was added to milk replacer at a feeding rate of 0.5 g/kg of body weight per head per day for the FAS and FAS+TT groups. Health exams were conducted twice weekly for 6 wk to diagnose BRD, navel inflammation, and diarrhea. Body weight, body condition score (BCS), and structural measurements were recorded at wk 0, 5, and 10. Average daily gain (ADG) was calculated for wk 1 to 5, wk 5 to 10, and wk 1 to 10. Health data were analyzed using logistic regression and are reported as relative risk. Body weight, BCS, and structural measurements were analyzed using repeated-measures ANOVA, and ADG was analyzed using a generalized linear model. There was no effect of FAS, TT, or their interaction on body weight or BCS. There was no effect of FAS on ADG wk 1 to 5, wk 5 to 10, or wk 1 to 10. There was a tendency for TT to decrease ADG from wk 1 to 5 but not from wk 5 to 10 or wk 1 to 10. There was a tendency for the interaction of FAS and TT to decrease ADG for wk 1 to 5 but not for wk 5 to 10 and wk 1 to 10. There was no effect of FAS, TT, or their interaction on the risk of BRD, diarrhea, or navel inflammation. We saw no effect of our interventions on calf health or growth. More research is needed to determine whether other factors, such as failure of passive transfer, poor ventilation, barren housing, and low milk allowance in the first few weeks after arrival, may have affected the efficacy of our interventions.

Iminosugars: Effects of Stereochemistry, Ring Size, and N-Substituents on Glucosidase Activities.

N-substituted iminosugar analogues are potent inhibitors of glucosidases and glycosyltransferases with broad therapeutic applications, such as treatment of diabetes and Gaucher disease, immunosuppressive activities, and antibacterial and antiviral effects against HIV, HPV, hepatitis C, bovine diarrhea (BVDV), Ebola (EBOV) and Marburg viruses (MARV), influenza, Zika, and dengue virus. Based on our previous work on functionalized isomeric 1,5-dideoxy-1,5-imino-D-gulitol (L-gulo-piperidines, with inverted configuration at C-2 and C-5 in respect to glucose or deoxynojirimycin (DNJ)) and 1,6-dideoxy-1,6-imino-D-mannitol (D-manno-azepane derivatives) cores N-linked to different sites of glucopyranose units, we continue our studies on these alternative iminosugars bearing simple N-alkyl chains instead of glucose to understand if these easily accessed scaffolds could preserve the inhibition profile of the corresponding glucose-based N-alkyl derivatives as DNJ cores found in miglustat and miglitol drugs. Thus, a small library of iminosugars (14 compounds) displaying different stereochemistry, ring size, and N-substitutions was successfully synthesized from a common precursor, D-mannitol, by utilizing an SN2 aminocyclization reaction via two isomeric bis-epoxides. The evaluation of the prospective inhibitors on glucosidases revealed that merely D-gluco-piperidine (miglitol, 41a) and L-ido-azepane (41b) DNJ-derivatives bearing the N-hydroxylethyl group showed inhibition towards α-glucosidase with IC50 41 µM and 138 µM, respectively, using DNJ as reference (IC50 134 µM). On the other hand, β-glucosidase inhibition was achieved for glucose-inverted configuration (C-2 and C-5) derivatives, as novel L-gulo-piperidine (27a) and D-manno-azepane (27b), preserving the N-butyl chain, with IC50 109 and 184 µM, respectively, comparable to miglustat with the same N-butyl substituent (40a, IC50 172 µM). Interestingly, the seven-membered ring L-ido-azepane (40b) displayed near twice the activity (IC50 80 µM) of the corresponding D-gluco-piperidine miglustat drug (40a). Furthermore, besides α-glucosidase inhibition, both miglitol (41a) and L-ido-azepane (41b) proved to be the strongest β-glucosidase inhibitors of the series with IC50 of 4 µM.

Development of PCR and Multiplex-PCR for Detection of Some Escherichia coli Virulence Genes from Bovine Neonates Diarrhea

Development of PCR and Multiplex-PCR for Detection of Some Escherichia coli Virulence Genes from Bovine Neonates Diarrhea

Synchronous Detection of BPV and BVDV with Duplex Taqman qPCR Method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrhea in dairy herds. BPV is a member of bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirus genus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes according to the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viral diarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the present study was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV. Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved 5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed in vitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, and then used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV. Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probe qRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were 2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplification conditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTR gene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. For clinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealing temperature was achieved in 43.2 ℃ fro duplex BPV and BPIV. Dynamic curves and standard curves were created following amplification of recombinant plasmids using the optimized duplex Taqman BPV and BVDV, with an amplification efficiency of 95.69%. Duplex Taqman qPCR could only detect DNA of BPV and cDNA of BVDV with a strong specificity. The detection limitation was as low as 2.0 × 102 copies/μL of pMD18-T-BPV plasmid and 2.0 × 101 copies/μL for pMD18-T-BVDV plasmid, respectively. Sensitivity of detection was 100-fold higher than conventional PCR. Duplex Taqman qPCR had excellent repeatability or stability with less than 1.2% of intra-assay and inter-assay. 35 and 47 positive feces samples were identified using duplex Taqman qPCR in comparison to 30 and 42 positives for universal PCR, respectively. Discussion: The bovine viral diarrhea virus (BVDV) is a key pathogenic factor in bovine diarrhea. Currently, few effective measures are available for the treatment or prevention for BVDV and BPV infections in animals. The technique was proven to be repeatable and linear over a range of at least 5 magnitudes, from 101 to 105 RNA/DNA copies, thus ensuring an accurate measurement of BPV DNA and BVDV RNA loads in clinical samples. In conclusion, a duplex Taqman qPCR was established for detecting simultaneously BPV and BVDV. Taqman qPCR method was rapid and specific assay. This assay was 100-fold sensitive than conventional PCR. It will be propitious to rapidly and differentially diagnose pathogens of viral diarrhea of dairy farms. Taqman qPCR method was rapid and specific assay and had a sensitivity of 2.0 copies/μL.

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate-binding domain (VP8*).

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate-binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus-cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*64-224 of canine rotavirus strain K9 interacts with N-acetylneuraminic and N-glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase-sensitive animal rotaviruses from pigs (CRW-8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N-glycolyl- over the N-acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.

Molecular characterization of rotaviruses isolated from calves with bovine neonatal diarrhea (BND) in Colombia

Bovine rotaviruses are one of the main agents involved in the presentation of Bovine Neonatal Diarrhea (BND), a disease that affects calves in the first month of life. Objective: The present study aimed to determine the types of bovine rotaviruses that affect dairy herds in the Sabana region of Bogota, Colombia. Materials and methods: 132 fecal samples were obtained from calves of less than five weeks of age and subjected to an ELISA test. MA104 cell cultures were infected with positive samples in order to isolate rotaviruses. The presence of the viral genome was confirmed by amplification and sequencing of a region of the viral VP7 protein-encoding gene. Results: Of the 132 samples, 26 (19, 7%) were ELISA-positive and nine samples were used for viral isolation. PCR amplification was achieved in all infected cultures. Sequencing showed homology of five samples to the G6 genotype. In addition, the presence of the G10 genotype was first determined for the country. Discussion: A greater presence of the G6 genotype from lineage V was found in the Sabana region of Bogota, showing a high prevalence in cattle and association with the presence of BND. The presence of the G10 genotype is a new report for the country and constitutes a new element of investigation in these viruses.

In Vivo Characterisation of Five Strains of Bovine Viral Diarrhoea Virus 1 (Subgenotype 1c).

Bovine viral diarrhoea virus 1 (BVDV-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.