Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers.

The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method

Evolution under different storage conditions of anomalous blue coloration of Mozzarella cheese intentionally contaminated with a pigment-producing strain of Pseudomonas fluorescens

Several widespread occurrences of anomalous blue coloration of Mozzarella cheese have been recorded in the United States and some European countries. Official laboratory analysis and health authorities have linked the occurrences to contamination of the processing water with strains of Pseudomonas fluorescens, although several experts questioned how to unequivocally link the blue color to the presence of the microorganism. To establish a method to determine whether a given Pseudomonas spp. strain is responsible for the defect and study the evolution of the coloration under different storage conditions, we developed an in vitro system for the evaluation of blue coloration of Mozzarella cheese intentionally contaminated with strains of P. fluorescens. The purpose of the system was to determine whether P.fluorescens strains, isolated from Mozzarella cheese with anomalous blue coloration, were able to reproduce the blue coloration under controlled experimental conditions. Thirty-six trials of experimental inoculation of Mozzarella cheese in different preservation liquids were conducted using various suspensions of P.fluorescens (P. fluorescens ATCC 13525, P.fluorescens CFBP 3150, and P. fluorescens 349 field strain isolated from blue-colored Mozzarella cheese) at different concentrations and incubated at different temperatures. Growth curves of all tested P.fluorescens strains demonstrated that after 3 d of incubation the concentration was generally >106 cfu/g of Mozzarella cheese incubated in either tryptic soy broth (control) or conditioning brine. Prolonged incubation for 5 d at either 20°C or 8°C led to concentrations up to 109 cfu/g of Mozzarella cheese incubated in tryptic soy broth and up to 108 cfu/g of Mozzarella cheese incubated in preservation liquid. All Mozzarella cheeses inoculated with the field strain of P. fluorescens, except those opened 1 h after packaging and stored at 8°C, showed the characteristic anomalous blue coloration, which appeared from 1 to 72 h after opening the packaging, and was proportional to colony count, duration of storage, and storage temperature. With the proposed system, which enabled a larger number of samples to be analyzed under controlled experimental conditions and a large amount of data to be generated in a short time, we described precisely how and under which conditions the presence of P. fluorescens in Mozzarella cheese is responsible for the anomalous blue coloration. The system will help producers intercept contaminated batches and help consumers avoid the conditions under which the defect can appear.Grants from Fondazione Cassa di Risparmio di Perugia (Italy) and from the Ministero degli Affari Esteri, Direzione Generale per la Promozione e Cooperazione Culturale (Rome, Italy),http://www.journals.elsevier.com/journal-of-dairy-sciencehb2017Paraclinical Science

The relationship between S. aureus and branched-chain amino acids content in composite cow milk

The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs—isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coe cient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p < 0.0001), 0.586 for leucine (p < 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.http://www.mdpi.com/journal/animalsam2020Paraclinical Science

Reduced paediatric hospitalizations for malaria and febrile illness patterns following implementation of community-based malaria control programme in rural Rwanda

<p>Abstract</p> <p>Background</p> <p>Malaria control is currently receiving significant international commitment. As part of this commitment, Rwanda has undertaken a two-pronged approach to combating malaria via mass distribution of long-lasting insecticidal-treated nets and distribution of antimalarial medications by community health workers. This study attempted to measure the impact of these interventions on paediatric hospitalizations for malaria and on laboratory markers of disease severity.</p> <p>Methods</p> <p>A retrospective analysis of hospital records pre- and post-community-based malaria control interventions at a district hospital in rural Rwanda was performed. The interventions took place in August 2006 in the region served by the hospital and consisted of mass insecticide treated net distribution and community health workers antimalarial medication disbursement. The study periods consisted of the December–February high transmission seasons pre- and post-rollout. The record review examined a total of 551 paediatric admissions to identify 1) laboratory-confirmed malaria, defined by thick smear examination, 2) suspected malaria, defined as fever and symptoms consistent with malaria in the absence of an alternate cause, and 3) all-cause admissions. To define the impact of the intervention on clinical markers of malaria disease, trends in admission peripheral parasitaemia and haemoglobin were analyzed. To define accuracy of clinical diagnoses, trends in proportions of malaria admissions which were microscopy-confirmed before and after the intervention were examined. Finally, to assess overall management of febrile illnesses antibiotic use was described.</p> <p>Results</p> <p>Of the 551 total admissions, 268 (48.6%) and 437 (79.3%) were attributable to laboratory-confirmed and suspected malaria, respectively. The absolute number of admissions due to suspected malaria was smaller during the post-intervention period (N = 150) relative to the pre-intervention period (N = 287), in spite of an increase in the absolute number of hospitalizations due to other causes during the post-intervention period. The percentage of suspected malaria admissions that were laboratory-confirmed was greater during the pre-intervention period (80.4%) relative to the post-intervention period (48.1%, prevalence ratio [PR]: 1.67; 95% CI: 1.39 – 2.02; chi-squared p-value < 0.0001). Among children admitted with laboratory-confirmed malaria, the risk of high parasitaemia was higher during the pre-intervention period relative to the post-intervention period (age-adjusted PR: 1.62; 95% CI: 1.11 – 2.38; chi-squared p-value = 0.004), and the risk of severe anaemia was more than twofold greater during the pre-intervention period (age-adjusted PR: 2.47; 95% CI: 0.84 – 7.24; chi-squared p-value = 0.08). Antibiotic use was common, with 70.7% of all children with clinical malaria and 86.4% of children with slide-negative malaria receiving antibacterial therapy.</p> <p>Conclusion</p> <p>This study suggests that both admissions for malaria and laboratory markers of clinical disease among children may be rapidly reduced following community-based malaria control efforts. Additionally, this study highlights the problem of over-diagnosis and over-treatment of malaria in malaria-endemic regions, especially as malaria prevalence falls. More accurate diagnosis and management of febrile illnesses is critically needed both now and as fever aetiologies change with further reductions in malaria.</p

How Does Institutional Change Coincide with Changes in the Quality of Life? An Exemplary Case Study

This paper provides a framework to assess correlations between the change of institutional functions (political centralization, plurality, rule of law, security of property, economic liberty, measured by 12 indicators) and improvements in human development (income, education, health) and violence limitations (conflict-related death tolls) to separate effective from ineffective institutional change. We apply this framework to a low-end institutional environment and provide a century case study of todays Democratic Republic of the Congo (DRC). Major results are threefold: first, we provide a thick description of institutional development in the Congo in a colonial and post-colonial and hence long-run setting; secondly, we identify periods of institutional change with distinctly different degrees of effectiveness; and thirdly, we are able to provide qualitative information on the questions of perspective (we follow a non-elitist approach), institutional connections, and timing of effects. Finally we propose extension of the framework, especially with respect to in-depth studies of critical transition periods, and its application to comparative case studies

Repayment behaviour in credit and savings cooperative societies

PurposeLike other developing countries, Rwandan rural credit market is repressed, shallow, segmented, inefficient and dual structured where both formal and informal financial systems operate side by side. While the later has been playing a predominant role, cooperative societies have emerged as an apt method of increasing the delivery of formal rural credit and savings facilities on sustainable and non‐exploitative terms albeit of financial imprudence stemming from poor credit repayment records. Thus, the purpose of this paper is to examine the factors contributing to credit repayment behaviour among the members of savings and credit cooperative societies in rural Rwanda.Design/methodology/approachBoth exploratory and descriptive designs are used for primary data collection on variables contributing to the repayment behaviour in savings and cooperative societies. Thereafter, a binary logistic regression empirical model is employed to estimate the contribution of each variable to credit repayment rate.FindingsThe results from the tested empirical model show that age, gender and size of the household, purpose for credit, interest rate charges and number of official visits to the credit societies, have a strong effect on loan repayment performance (statistically significant at p&lt;0.05) whereas size of credit disbursed, credit processing and disbursing time, borrowers' market place and income transfer from relatives and friends are more or less statistically significant at p&lt;0.20 level. The remaining factors have logical and explainable sings but are not statistically significant.Research limitations/implicationsThe primary limitation of this study is the scope and size of its sample as well as absence of income factor as one of important variable influencing repayment behaviour. These limitations may have an effect on the lending policy of the cooperative banks.Originality/valueAn understanding of the socio‐economic factors affecting repayment behaviour of rural clients is essential for the outreach and sustainability of the mushrooming cooperative societies in the country. Hence, this paper contributes to the empirical literature on the provision of rural financial services in African countries south of Sahara and Rwanda in particular.</jats:sec

Repayment behaviour in credit and savings cooperative societies: Empirical and theoretical evidence from rural Rwanda

Purpose – Like other developing countries, Rwandan rural credit market is repressed, shallow, segmented, inefficient and dual structured where both formal and informal financial systems operate side by side. While the later has been playing a predominant role, cooperative societies have emerged as an apt method of increasing the delivery of formal rural credit and savings facilities on sustainable and non-exploitative terms albeit of financial imprudence stemming from poor credit repayment records. Thus, the purpose of this paper is to examine the factors contributing to credit repayment behaviour among the members of savings and credit cooperative societies in rural Rwanda. Design/methodology/approach – Both exploratory and descriptive designs are used for primary data collection on variables contributing to the repayment behaviour in savings and cooperative societies. Thereafter, a binary logistic regression empirical model is employed to estimate the contribution of each variable to credit repayment rate. Findings – The results from the tested empirical model show that age, gender and size of the household, purpose for credit, interest rate charges and number of official visits to the credit societies, have a strong effect on loan repayment performance (statistically significant at pCo-operative societies, Credit, Loans, Rural areas, Rwanda

Phenotypic and Genotypic Characterization of Verotoxin-Producing Escherichia coli O103:H2 Isolates from Cattle and Humans▿

Characterization of important non-O157 verotoxin-producing Escherichia coli (VTEC) has lagged considerably behind that of O157:H7 strains. This study characterized 91 VTEC O103:H2 strains from bovine and human sources and of North American and European origins by virulence or putative virulence genes, pulsed-field gel electrophoresis (PFGE) patterns, plasmid profiles, antimicrobial resistance, and colicin production. All strains were positive for vt1 and eae-ɛ; 97% were positive for ehxA; and all were negative for hlyA. Two strains carried vt2. There were 66 PFGE patterns grouped in six clusters, and there were 25 different plasmid profiles. Plasmid-encoded katP and etp genes were significantly more frequent in European than in North American human strains. The distribution of selected phenotypes was as follows: enterohemorrhagic E. coli (EHEC) hemolysin, 95%; colicin production, 38%; antimicrobial resistance, 58%. All the strains were negative for the alpha-hemolytic phenotype. In conclusion, the VTEC O103:H2 strains were diverse, as shown by PFGE, plasmid profiles, virulence markers, and antimicrobial resistance patterns, and all strains showed an EHEC hemolytic phenotype instead of the alpha-hemolytic phenotype that has been shown previously