Dairy Cattle Feces Research Articles

Prevalence and antimicrobial susceptibility profile of Salmonella isolated from vegetable farms fertilized with animal manure in Addis Ababa Ethiopia

The resistance of foodborne pathogens to antimicrobial agents is a potential danger to human health. Hence, establishing the status of good agricultural practices (GAPs) and the antimicrobial susceptibility of major foodborne pathogens has a significant programmatic implication in planning interventions. The objective of this study was to assess the gap in attaining GAP and estimate the prevalence and antimicrobial susceptibility profile of Salmonella in vegetable farms fertilized with animal manure in Addis Ababa, Ethiopia. A total of 81 vegetable farms from four sub-cities in Addis Ababa were visited, and 1119 samples were collected: soil (n = 271), manure (n = 375), vegetables (n = 398), and dairy cattle feces (n = 75). Additional data were collected using a structured questionnaire. Isolation of Salmonella was done using standard microbiology techniques and antimicrobial susceptibility testing was conducted using disk diffusion assays. Carriage for antimicrobial resistance genes was tested using polymerase chain reaction (PCR). Among the 81 vegetable farms visited, 24.7% used animal manure without any treatment, 27.2% used properly stored animal manure and 80.2% were easily accessible to animals. The prevalence of Salmonella was 2.3% at the sample level, 17.3% at the vegetable farm level, and 2.5% in vegetables. The highest rate of resistance was recorded for streptomycin, 80.7% (21 of 26), followed by kanamycin, 65.4% (17 of 26), and gentamicin, 61.5% (16 of 26). Multidrug resistance was detected in 61.5% of the Salmonella isolates. Vegetable farms have a gap in attaining GAPs, which could contribute to increased contamination and the transfer of antimicrobial resistance to the vegetables. The application of GAPs, including proper preparation of compost and the appropriate use of antimicrobials in veterinary practices, are recommended to reduce the emergence and spread of antimicrobial resistance.

A newly identified IncY plasmid from multi-drug-resistant Escherichia coli isolated from dairy cattle feces in Poland.

This study reveals the identification of new strains of antibiotic-resistant Escherichia coli in cattle manure from a dairy farm in Poland, offering critical insights into the spread of drug resistance. Through whole-genome sequencing, researchers discovered novel plasmids within these bacteria, which carry genes resistant to multiple antibiotics. These findings are particularly alarming, as these plasmids can transfer between different bacterial species, potentially escalating the spread of antibiotic resistance. This research underscores the vital connection between the health of humans, animals, and the environment, emphasizing the concept of One Health. It points to the critical need for global vigilance and strategies to curb the proliferation of antibiotic resistance. By showcasing the presence of these strains and their advanced resistance mechanisms, the study calls for enhanced surveillance and preventive actions in both agricultural practices and healthcare settings to address the imminent challenge of antibiotic-resistant bacteria.

Microdiversity of Salmonella Kentucky During Long-Term Colonization of a Dairy Herd.

Salmonella enterica subsp. enterica serovar Kentucky was repeatedly isolated from a commercial dairy herd that was enrolled in a longitudinal study where feces of asymptomatic dairy cattle were sampled intensively over an 8-year period. The genomes of 5 Salmonella Kentucky isolates recovered from the farm 2 years before the onset of the long-term colonization event and 13 isolates collected during the period of endemicity were sequenced. A phylogenetic analysis inferred that the Salmonella Kentucky strains from the farm were distinct from poultry strains collected from the same region, and three subclades (K, A1, and A2) were identified among the farm isolates, each appearing at different times during the study. Based on the phylogenetic analysis, three separate lineages of highly similar Salmonella Kentucky were present in succession on the farm. Genomic heterogeneity between the clades helped identify regions, most notably transcriptional regulators, of the Salmonella Kentucky genome that may be involved in competition among highly similar strains. Notably, a region annotated as a hemolysin expression modulating protein (Hha) was identified in a putative plasmid region of strains that colonized a large portion of cows in the herd, suggesting that it may play a role in asymptomatic persistence within the bovine intestine. A cell culture assay of isolates from the three clades with bovine epithelial cells demonstrated a trend of decreased invasiveness of Salmonella Kentucky isolates over time, suggesting that clade-specific interactions with the animals on the farm may have played a role in the dynamics of strain succession. Results of this analysis further demonstrate an underappreciated level of genomic diversity within strains of the same Salmonella serovar, particularly those isolated during a long-term period of asymptomatic colonization within a single dairy herd.

Antibiogram of Escherichia coli Isolated from Dairy Cattle and in-Contact Humans in Selected Areas of Central Ethiopia.

Antimicrobial resistance (AMR) is a global threat to public and animal health. Escherichia coli is considered an indicator organism for monitoring AMR among gram-negative Enterobacteriaceae in humans and animals. The current study aims to assess the antibiogram profile of E. coli isolated from dairy cattle and in-contact humans in central Ethiopia and to identify risk factors associated with multidrug resistance (MDR). A cross-sectional study was conducted in which 58 farms were recruited from selected districts of central Ethiopia. E. coli was isolated using standard bacteriological techniques. A total of 200 representative isolates (140 from cattle and 60 from humans in contact) were randomly selected and tested for susceptibility to a panel of 13 antimicrobials using the Kirby-Bauer disc diffusion assay. The highest rate of resistance was observed for sulfamethoxazole+trimethoprim (58.6%, 82/140) and amoxicillin+clavulanic acid (70.0%, 42/60) among E. coli isolates from cattle and hmans, respectively. In contrast, resistance rates in isolates from in contact humans with the cattle were 30%, 33.3%, and 66.7%, respectively. Resistance to tetracycline (p=0.02), streptomycin (p=0.03), and sulfamethoxazole+trimethoprim (p=0.007) was significantly high in E. coli isolated from cattle on commercial dairy farms than in those isolated from cattle on smallholder farms. There was no significant difference (p>0.05) in the rate of resistance between E. coli isolated from in contact humans with smallholder and commercial dairy farms. Antimicrobial use for treatment purpose (p=0.04) and non-compliance with the drug withdrawal period (p=0.03) were significantly associated with the farm-level occurrence of MDR. A high rate of resistance was detected in E. coli isolated from the feces of dairy cattle and in-contact humans. This necessitates an effective intervention through a one-health approach and further molecular studies are required to establish source attribution.

Diarrheagenic Escherichia coli with Multidrug Resistance in Cattle from Mexico

Mexico is an important producer/exporter of cattle and cattle products. In the last decade, an increase in antibiotic resistance in E. coli pathotype strains from livestock environments has been reported. This study aimed to determine the prevalence and antibiotic resistance profiles of E. coli pathotype strains from the feces of beef or dairy cattle reared in the states of Aguascalientes (AG, central) and Nuevo Leon (NL, northeastern) in Mexico. One hundred and ten fecal samples were collected (beef cattle-AG = 30; dairy cattle-AG = 20; beef cattle-NL = 30; dairy cattle-NL = 30). From these, E. coli was isolated using selective/differential media and confirmed on chromogenic media. Multiplex PCR was used to identify diarrheagenic E. coli, and the Kirby–Bauer technique was used to determine the antimicrobial susceptibilities. All the animals harbored E. coli, and pathotypes were found in 34 animals from both, beef and dairy cattle, mainly from Aguascalientes. Of the positive samples, 31 harbored a single E. coli pathotype, whereas three samples harbored two different pathotypes; EHEC was the most prevalent, followed by EPEC, ETEC, and EIEC or the combination of two of them in some samples. Most pathotype strains (19/37) were isolated from beef cattle. Neither the animals’ productive purpose (beef or dairy cattle) (r = 0.155) nor the geographic regions (Aguascalientes or Nuevo Leon) (r = −0.066) had a strong positive correlation with the number of E. coli pathotype strains. However, animals reared in Aguascalientes had up to 8.5-fold higher risk of harboring E. coli pathotype strains than those reared in Nuevo Leon. All pathotype strains were resistant to erythromycin, tetracycline, and trimethoprim/sulfamethoxazole, and all dairy cattle pathotype strains were further resistant to five β-lactams (χ2, P = 0.017). The existence of these pathotypes and multidrug-resistant pathogens in the food chain is a risk to public health.

Analysis of Diversity and Distribution of Antibiotic Resistance Genes in Holstein Friesian Dairy Cattle Feces by Shotgun Metagenomic Approach

Analysis of Diversity and Distribution of Antibiotic Resistance Genes in Holstein Friesian Dairy Cattle Feces by Shotgun Metagenomic Approach

Application of Near-Infrared Reflectance Spectroscopy for Predicting Chemical Composition of Feces in Holstein Dairy Cows and Calves.

Traditional methods for determining the chemical composition of cattle feces are uneconomical. In contrast, near-infrared reflectance spectroscopy (NIRS) has emerged as a successful technique for assessing chemical compositions. Therefore, in this study, the feasibility of NIRS in terms of predicting fecal chemical composition was explored. Cattle fecal samples were subjected to chemical analysis using conventional wet chemistry techniques and a NIRS spectrometer. The resulting fecal spectra were used to construct predictive equations to estimate the chemical composition of the feces in both cows and calves. The coefficients of determination for calibration (RSQ) were employed to evaluate the calibration of the predictive equations. Calibration results for cows (dry matter [DM], RSQ = 0.98; crude protein [CP], RSQ = 0.93; ether extract [EE], RSQ = 0.91; neutral detergent fiber [NDF], RSQ = 0.82; acid detergent fiber [ADF], RSQ = 0.89; ash, RSQ = 0.84) and calves (DM, RSQ = 0.92; CP, RSQ = 0.89; EE, RSQ = 0.77; NDF, RSQ = 0.76; ADF, RSQ = 0.92; ash, RSQ = 0.97) demonstrated that NIRS is a cost-effective and efficient alternative for assessing the chemical composition of dairy cattle feces. This provides a new method for rapidly predicting fecal chemical content in cows and calves.

Predicting orthophosphate in feces and manure from dairy cattle

Dairy cattle excreta are a valuable source of orthophosphate (Ortho-P), an inorganic form of phosphorus (P) that is readily available for microorganisms, plant growth, and development. There is, however, a growing environmental concern about the potential negative environmental impact of excessive amounts of Ortho-P excretion, which can lead to the eutrophication of water bodies. As a result, the development of mathematical equations to quantify and manage Ortho-P excretion on dairy farms could prove valuable for environmental sustainability. This study aimed to use literature data to develop empirical predictions for Ortho-P (g/kg dry matter [DM]) excretion using total P (TP [g/kg DM]) content of dairy cattle feces (Ortho-Pf) and manure (Ortho-Pm). Data sets from studies that evaluated and characterized the different forms of P in feces and manure from dairy cattle were compiled. After outlier exclusion, the final retained database for feces included 37 treatment means from 4 published papers while the manure comprised 23 treatment means from 7 published papers. A linear-mixed model was used to develop the predictive equations, incorporating the random effect of the study. A leave-one-out cross-validation procedure was used to evaluate the predictive ability of the developed models, whereby studies were regarded as folds. The fecal equation was determined as Ortho-Pf (g/kg DM) = −2.447 (0.572) + 0.966 (0.083) × TP (g/kg DM) (R2 = 0.79) and resulted in a root mean square prediction error as a percentage of mean observed value (RMSPE, %) of 32.8% and error due to random sources of 97.6%. Additionally, the manure equation was determined as Ortho-Pm (g/kg) = −0.204 (0.446) + 0.590 (0.065) × TP (g/kg) (R2 = 0.77) and had an RMSPE of 43.3% with a random error of 93.9%. Both models revealed minimal mean and slope biases on feces and manure data. Findings suggest that these sets of equations can be used to estimate excreted Ortho-P from total excreted P, helping nutritionists and farmers to understand the impact of dietary P changes on the environment. Further, these equations can be incorporated into extant models such as the Cornell Net Carbohydrate and Protein System (CNCPS) to aid in understanding and mitigating P and Ortho-P excretion from dairy cattle and to clarify the portion of P that migrates more rapidly into watersheds.

Survival of bovine digital dermatitis treponemes in conditions relevant to the host and farm environment

ObjectivesBovine digital dermatitis (BDD), a painful infectious foot disease in dairy cattle, endemic in many countries worldwide, causes substantial economic and welfare impacts. Treponema spp. are considered key to BDD pathogenesis. To aid infection reservoir identification and control measure development, survival of BDD treponemes was investigated in different temperatures (4, 12, 20, 37, 45 and 60 °C), pH values (5–9.0), dairy cattle faeces and bedding types: straw shavings, sand, sand containing 5% lime (w/w) and recycled manure solids (RMS). MethodsA turbidity microplate methodology was adapted to measure pH impact on growth. Survival of BDD treponemes for the different conditions were assessed by sub-cultures of microcosms over different time points. ResultsBDD treponemes remained viable between 4 and 37 °C and pH 5.5 and 9.0 under anaerobic conditions. In sterile faecal microcosms, incubated aerobically at 12 °C, BDD treponemes remained viable for a median of 1 day (15 min - 6 day range). Variation in duration of survival and ability to grow was observed between phylogroups and strains. In aerobic microcosms, T. phagedenis T320A remained viable for the full 7 days in sand, 6 days in sawdust, 5 days in RMS, but was not viable after 15 min in straw or sand containing 5% (w/w) lime. ConclusionsTreponeme survival conditions identified here should enhance future BDD infection reservoir surveys and enable control measures. Of note, straw or sand containing 5% (w/w) lime should be assessed in BDD field trials. Finally, these data indicate BDD treponemes exhibit characteristics of facultative anaerobes.

Incidence of Extended Spectrum Beta-Lactamase (ESBL) of Escherichia Coli Isolated from the Feces of Dairy Cattles in Blitar

Antibiotic resistance (AMR) has caused a serious global threat to human health, animal health and food security. The antibiotic resistance problem, especially in Gram-negative bacteria, is increasingly widespread in various areas and has increased mortality, morbidity, and incidence. This study aims to determine the incidence of ESBL E. coli and the AMR and MDR properties of E.coli isolated from the feces of dairy cattles in Blitar, which have not been reported. This research was conducted from June to September 2022 with 60 samples of dairy cattle feces in Blitar. Samples were isolated and identified until confirmed that 55 (91.6%) samples were E. coli. All samples showed AMR character with 100% streptomycin, 87.27% chloramphenicol, 85.45% ampicillin, 72.73% cefotaxime, 40% tetracycline, 21.82% ciprofloxacin, and 14.55% sulfamethoxazole-trimethoprim. Forty-nine samples were MDR, with 10.9% resistant in three groups, 34.5% in four groups, 29.1% in five groups, 10.9% in six groups, and 3.6% in seven groups. MDR E. coli isolates were then confirmed for ESBL using the DDST test. The incidence of ESBL-producing E. coli from dairy cattle feces in Blitar was 40% (22 isolates). The results of this study can be a warning in the human health, animal health and food security.

Determination of Non-Digestible Parts in Dairy Cattle Feces Using U-NET and F-CRN Architectures.

Deep learning algorithms can now be used to identify, locate, and count items in an image thanks to advancements in image processing technology. The successful application of image processing technology in different fields has attracted much attention in the field of agriculture in recent years. This research was done to ascertain the number of indigestible cereal grains in animal feces using an image processing method. In this study, a regression-based way of object counting was used to predict the number of cereal grains in the feces. For this purpose, we have developed two different neural network architectures based upon Fully Convolutional Regression Networks (FCRN) and U-Net. The images used in the study were obtained from three different dairy cows enterprises operating in Nigde Province. The dataset consists of the 277 distinct dropping images of dairy cows in the farm. According to findings of the study, both models yielded quite acceptable prediction accuracy with U-Net providing slightly better prediction with a MAE value of 16.69 in the best case, compared to 23.65 MAE value of FCRN with the same batch.

Time since faecal deposition influences mobilisation of culturable E. coli and intestinal enterococci from deer, goose and dairy cow faeces.

Mobilisation is a term used to describe the supply of a pollutant from its environmental source, e.g., soil or faeces, into a hydrological transfer pathway. The overarching aim of this study was to determine, using a laboratory-based approach, whether faecal indicator bacteria (FIB) are hydrologically mobilised in different quantities from a typical agricultural, wildlife and wildfowl source, namely dairy cattle, red deer and greylag goose faeces. The mobilisation of FIB from fresh and ageing faeces under two contrasting temperatures was determined, with significant differences in the concentrations of both E. coli and intestinal enterococci lost from all faecal sources. FIB mobilisation from these faecal matrices followed the order of dairy cow > goose > deer (greatest to least, expressed as a proportion of the total FIB present). Significant changes in mobilisation rates from faecal sources over time were also recorded and this was influenced by the temperature at which the faecal material had aged over the course of the 12-day study. Characterising how indicators of waterborne pathogens are mobilised in the environment is of fundamental importance to inform models and risk assessments and develop effective strategies for reducing microbial pollution in catchment drainage waters and associated downstream impacts. Our findings add quantitative evidence to support the understanding of FIB mobilisation potential from three important faecal sources in the environment.

Prevalence of Multidrug-Resistant CTX-M Extended Spectrum Beta-Lactamase-Producing Escherichia coli From Different Bovine Faeces in China.

CTX-M extended spectrum beta-lactamase-producing Escherichia coli cause severe health hazards in livestock breeding. To date, little is known about antibiotic resistance differences among bacterial isolates from yaks, cows, and beef cattle; therefore, the aims of this study were to analyse the prevalence of CTX-M-producing E. coli in yak, beef cattle, and dairy cattle feces from different provinces in China. A total of 790 fecal samples from yaks, beef cattle, and dairy cows were used. Among all the samples, 523 non duplicate E. coli isolates were identified, and 29.6% of samples harbored CTX-M producers. The results showed that these E. coli strains harbored 15 clusters of CTX-M genes: CTX-M-79, CTX-M-55, CTX-M-15, CTX-M-14, CTX-M-28, CTX-M-179, CTX-M-65, CTX-M-24, CTX-M-27, CTX-M-102, CTX-M-105, CTX-M-173, CTX-M-238, CTX-M-196, and CTX-M-10. The dominant resistance genes were CTX-M-15, CTX-M-14, and CTX-M-55. Moreover, the distribution of CTX-M genes was related to geographical region. Based on the above findings, we reasoned that bovines are potential reservoirs of antibiotic resistance, and this problem should be given adequate attention.

The epidemiology of AmpC-producing Escherichia coli isolated from dairy cattle faeces on pasture-fed farms.

IntroductionAntibiotic use, particularly amoxicillin-clavulanic acid in dairy farming, has been associated with an increased incidence of AmpC-hyperproducing Escherichia coli .Gap statementThere is limited information on the incidence of AmpC-hyperproducing E. coli from seasonal pasture-fed dairy farms.AimWe undertook a New Zealand wide cross-sectional study to determine the prevalence of AmpC-producing E. coli carried by dairy cattle.MethodologyPaddock faeces were sampled from twenty-six dairy farms and were processed for the selective growth of both extended-spectrum beta-lactamase (ESBL)- and AmpC-producing E. coli . Whole genome sequence analysis was carried out on 35 AmpC-producing E. coli .ResultsNo ESBL- or plasmid mediated AmpC-producing E. coli were detected, but seven farms were positive for chromosomal mediated AmpC-hyperproducing E. coli . These seven farms were associated with a higher usage of injectable amoxicillin antibiotics. Whole genome sequence analysis of the AmpC-producing E. coli demonstrated that the same strain (<3 SNPs difference) of E. coli ST5729 was shared between cows on a single farm. Similarly, the same strain (≤15 SNPs difference) of E. coli ST8977 was shared across two farms (separated by approximately 425 km).ConclusionThese results infer that both cow-to-cow and farm-to-farm transmission of AmpC-producing E. coli has occurred.

Quantification of zinc concentrations in serum, milk and faeces of dairy cattle as a measure of effective zinc supplementation for management of facial eczema

Aims: To investigate the relationship between Zn concentrations in serum and those in milk or faeces, and to assess the ability of the Zn concentrations in milk, serum and faeces to predict intake of ZnO in dairy cattle. Method Seventy cows from one commercial farm in the Waikato region of New Zealand received one of seven dose rates (0, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 g/100 kg bodyweight (BW)) of ZnO given by oral drench, every morning, for 7 consecutive days. Every afternoon, milk and blood samples were collected from all cows. Free-catch faecal samples were collected during the afternoon milking on 3 days throughout the trial. Linear mixed models were used to assess the relationship between the concentration of Zn in serum and that in milk, and in faeces, respectively, and the relationship between dose rate of ZnO and concentrations of Zn in serum, faeces and milk, respectively. Receiver operating characteristic curve analysis was used to determine the ability of the Zn concentration in serum, milk and faeces to predict that a cow had been treated with a dose of ZnO ≥2.5 g/100 kg, the industry-recommended dose rate needed to protect against facial eczema. Results: A 1-µmol/L increase in Zn concentration in milk was associated with a 0.14 (95% CI=0.11–0.17) µmol/L increase in Zn concentration in serum. Zn concentration in faeces was scaled by its SD; a 1 SD increase was associated with a 1.83 (95% CI=0.54–3.12) µmol/L increase in zinc concentration in serum. Zn concentrations in serum and faeces increased with increasing dose rates of ZnO. No differences in Zn concentrations in milk were noted between animals dosed with 1.5–3.5 g ZnO/100 kg BW, inclusive. At the optimal threshold for Zn concentration in serum, 22 µmol/L, the sensitivity was 0.76 (95% CI=0.69–0.82) and specificity 0.85 (95% CI=0.80–0.89). For the concentration of Zn in faeces, the optimal threshold was defined as 17.36 mmol/kg, with a corresponding sensitivity of 0.84 (95% CI=0.84–0.85) and specificity of 0.85 (95% CI=0.73–0.94). At the optimal threshold for the Zn concentration in milk (76.6 µmol/L), the sensitivity was lower than the other two sample types at 0.59 (95% CI=0.52–0.67), but with a similar specificity of 0.84 (95% CI=0.79–0.88). Conclusions and clinical relevance: The concentration of Zn in milk shows promise as an initial screening test to identify dairy farms that do not provide adequate zinc to provide protection against FE.

Antibiotic resistance among Escherichia coli and Salmonella isolated from dairy cattle feces in Texas.

In several developing countries, studies on antimicrobial resistance among bacteria from food animals are rare mostly because of under-resourced laboratories. The objective of this study was to develop and field-test a low cost protocol to estimate the isolate- and sample-level prevalence of resistance to critically important antibiotics among Escherichia coli and Salmonella isolated from dairy cattle feces. Using a predesigned protocol, fecal samples were collected to isolate non-type-specific E. coli and Salmonella using selective media without antibiotic supplements. Besides, samples were screened for E. coli and Salmonella isolates not susceptible to third-generation cephalosporins and quinolones using selective media supplemented with cefotaxime (1.0 μg/mL) and ciprofloxacine (0.5 μg/mL), respectively. All bacterial isolates were further tested for antibiotic susceptibility using disk diffusion. Bacterial isolates not susceptible to third-generation cephalosporins were tested for extended spectrum beta-lactamase (ESBL) phenotype using the combination disk test. Molecular methods were performed on selected bacterial isolates to identify and distinguish genetic determinants associated with the observed phenotypes. Among 85 non-type-specific E. coli isolated from MacConkey agar without antibiotics, the isolate-level prevalence of resistance to tetracycline was the highest (8.2%). Among 37 E. coli recovered from MacConkey agar with cefotaxime, 56.8% were resistant ceftriaxone. Among 22 E. coli isolates recovered from MacConkey agar with ciprofloxacin, 77.3% and 54.5% were resistant to nalidixic acid and ciprofloxacin, respectively. Sixteen Salmonella were isolated and only one demonstrated any resistance (i.e., single resistance to streptomycin). Among E. coli isolates not susceptible to ceftriaxone, an AmpC phenotype was more common than an ESBL phenotype (29 versus 10 isolates, respectively). Whole genome sequencing showed that phenotypic profiles of antibiotic resistance detected were generally substantiated by genotypic profiles. The tested protocol is suited to detecting and estimating prevalence of antimicrobial resistance in bacteria isolated from food animal feces in resource-limited laboratories in the developing world.

Detection of Mycobacterium avium sub sp. paratuberculosis (MAP) by PCR in the faeces of dairy cattle of Chitwan, Nepal

Johne's disease or Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic granulomatous enteritis with decrease in production resulting huge economic losses with high negative impact on the livestock industry. Diagnosis of MAP is difficult due to lack of characteristics clinical signs, prolong incubation period in cultivation of MAP, and non-specific results in diagnostic tests. To the best of our knowledge there is no report on faecal culture and molecular detection of MAP in dairy cattle of Nepal. The main objective of this research was to access the herd level prevalence of Johne’s disease in the representative dairy farms of Chitwan district with the use of modern techniques as faecal polymerase chain reaction (fPCR) to know the MAP distribution in dairy cattle. A total of 265 individual dairy cattle faeces sample were collected during February 2017 to January 2018 from dairy farms of three different geographical location of Chitwan district, Nepal. Faeces were decontaminated and subjected for faecal culture as well as fPCR to have molecular detection of MAP. Findings revealed that bio-load of MAP in dairy cattle were 13.57% by faecal culture, and 16.59% by fPCR detection method. The overall prevalence of MAP in dairy cattle was detected as 16.59 % by fPCR. Likewise, IS900 PCR assay proved to be a more sensitive and reliable test than faecal culture for the detection of MAP in faecal sample of clinically suspected dairy cattle as the PCR assay was able to detect significantly (p &lt; 0.01) more positive cases than faecal culture. Findings of this study suggests that IS900-PCR-based detection of MAP could be used as a potential diagnostic tool for rapid and effective Johne’s disease (JD) surveillance as compared with faecal culture detection method due to its advantage for JD control programs by reducing the time of definitive diagnosis from several months to a few days. This is the first molecular level of diagnostic research performed and reporting of MAP in dairy cattle of Nepal. These results will be useful in designing suitable disease control strategy for livestock industry.

A Comparison of Two DNA Metagenomic Bioinformatic Pipelines While Evaluating the Microbial Diversity in Feces of Tanzanian Small Holder Dairy Cattle.

Analysis of shotgun metagenomic data generated from next generation sequencing platforms can be done through a variety of bioinformatic pipelines. These pipelines employ different sets of sophisticated bioinformatics algorithms which may affect the results of this analysis. In this study, we compared two commonly used pipelines for shotgun metagenomic analysis: MG-RAST and Kraken 2, in terms of taxonomic classification, diversity analysis, and usability using their primarily default parameters. Overall, the two pipelines detected similar abundance distributions in the three most abundant taxa Proteobacteria, Firmicutes, and Bacteroidetes. Within bacterial domain, 497 genera were identified by both pipelines, while an additional 694 and 98 genera were solely identified by Kraken 2 and MG-RAST, respectively. 933 species were detected by the two algorithms. Kraken 2 solely detected 3550 species, while MG-RAST identified 557 species uniquely. For archaea, Kraken 2 generated 105 and 236 genera and species, respectively, while MG-RAST detected 60 genera and 88 species. 54 genera and 72 species were commonly detected by the two methods. Kraken 2 had a quicker analysis time (~4 hours) while MG-RAST took approximately 2 days per sample. This study revealed that Kraken 2 and MG-RAST generate comparable results and that a reliable high-level overview of sample is generated irrespective of the pipeline selected. However, Kraken 2 generated a more accurate taxonomic identification given the higher number of “Unclassified” reads in MG-RAST. The observed variations at the genus level show that a main restriction is using different databases for classification of the metagenomic data. The results of this research indicate that a more inclusive and representative classification of microbiomes may be achieved through creation of the combined pipelines.

Molecular profiling and antimicrobial susceptibility of Escherichia coli O157:H7 isolated in Bulgaria

The purpose of this study was to detect the presence of shiga-toxin producing Escherichia coli (STEC) in faeces of healthy dairy cattle and to determine the sensitivity of isolates to several anti­microbial drugs. A total of 1,104 anal swab samples originating from 28 cattle farms were examined. After the primary identification, 30 strains were found to belong to serogroup О157. By means of conventional multiplex PCR, isolates were screened for presence of resistance genes stx1, stx2 and eaeА. Twenty-nine strains possesses amplicons with a size corresponding to genes stx2 and eaeA, one had amplicons also for the stx1 gene and one lacked amplicons of all three genes. Twenty-eight strains demonstrated amplicons equivalent to gene H7. The results from phenotype analysis of resistance showed preserved sensitivity to ceftriaxone, ceftazidime, cefotaxime, cephalothin, streptomycin, gentamicin, tetracycline, enrofloxacin and combinations sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Sensitivity to ampicillin was relatively preserved, although at a lower extent.

Prevalence of Listeria Species in Dairy Cows and Pregnant Women with Reference to Virulotyping of Listeria monocytogenes in Egypt

This study was conducted to determine the prevalence of Listeria spp . in milk and feces of dairy cows as well as stool of pregnant women in Sharkia and Dakahlia Governorates, Egypt. Three hundred and fifty samples including; normal raw milk (n=200), mastitis milk (n=50), feces of dairy cattle (n=50) and stool of pregnant women (n=50) were randomly collected. Listeria spp. were isolated on Oxford agar and then subjected to biochemical and molecular identification. The overall isolation rate of Listeria spp. was 16%. The prevalence rates of Listeria monocytogenes were 8%, 4%, 2% and 4% in feces of dairy cows, normal milk, mastitis milk and stool of pregnant women, respectively. The prevalence of L. ivanovii and L. welshimeri in milk of dairy cows were 6% and 4%, respectively. L. monocytogenes isolates were molecularly confirmed to the species level by amplification of 16S rRNA gene. The distribution of internalin A (inlA) and internalin B (inlB) was determined in 15 identified L. monocytogenes isolates. The overall prevalence of inlA gene and inlB gene in L. monocytogenes isolates from different sources were 80% and 40%, respectively. Two L. monocytogenes isolates from normal raw milk and feces of dairy cows didn’t harbor both inlA and inlB genes. Listeria spp. isolated from normal milk, feces, mastitis milk and stool of pregnant women, potentially can cause human illness and abortion in pregnant women. Internalin (A&B) genes are considered the best indicator for virulence determination of L. monocytogenes isolated from different sources.