A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.Keywords: Environmental, DNA extraction, Feces, qPCR, Litter, Microbiomics, Molecular biology, Semi-automated, PoultryKeywords: Environmental, DNA extraction, Feces, qPCR, Litter, Microbiomics, Molecular biology, Semi-automated, Poultr

The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain

The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n = 45) and group B S. Heidelberg (n = 11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.Keywords: S. Heidelberg, Scalder water, Multidrug resistanc

Associations between Mycobacterium avium subsp. paratuberculosis antibodies in bulk tank milk, season of sampling and protocols for managing infected cows

BACKGROUND: The objective of this study was to identify associations between the concentration of Mycobacterium avium subsp. paratuberculosis (MAP) antibodies in bulk milk and potential risk factors in herd management and herd characteristics, explaining high MAP antibody titers in milk. An extensive questionnaire was administered to 292 organic and conventional dairy farms from New York, Wisconsin and Oregon. Bulk milk samples were taken from each farm for MAP enzyme-linked immunosorbent assay (ELISA). A general linear model was constructed with MAP ELISA value as the outcome variable and the management factors and herd characteristics as independent variables, while at the same time controlling for the study design variables of state, herd size, and production system (organic or conventional). High bulk tank MAP ELISA value may be due to either a high prevalence of MAP in a herd with many cows contributing to the antibody titer or due to a few infected cows that produce large quantities of antibodies. RESULTS: Results of the regression models indicated that bulk milk ELISA value was associated with season of sampling and the presence or absence of protocols for managing MAP-positive cows. The concentration of MAP antibodies in bulk milk varied seasonally with a peak in the summer and low concentrations in the winter months. When compared to farms that had never observed clinical Johne’s disease, keeping MAP-positive cows or only culling them after a period of delay was associated with an increase in optical density. CONCLUSIONS: The seasonal variation in MAP antibody titers, with a peak in the summer, may be due to a seasonal increase in MAP-bacterial load. Additionally, seasonal calving practices may contribute to seasonal fluctuations in MAP antibody titers in bulk tank milk. Keeping MAP-positive cows increases the antibody titer in bulk milk, likely due to direct antibody production in the infected cow and indirect triggering of antibody production in herdmates.Keywords: Antibodies, ELISA, Cattle, Mycobacteirum avium subsp. paratuberculosis, Bulk-tank milkKeywords: Antibodies, ELISA, Cattle, Mycobacteirum avium subsp. paratuberculosis, Bulk-tank mil

Bacteriological etiology and treatment of mastitis in Finnish dairy herds

Background: The Finnish dairy herd recording system maintains production and health records of cows and herds. Veterinarians and farmers register veterinary treatments in the system. Milk samples for microbiological analysis are routinely taken from mastitic cows. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes most samples using real-time PCR. This study addressed pathogen-specific microbiological data and treatment and culling records, in combination with cow and herd characteristics, from the Finnish dairy herd recording system during 2010-2012. Results: The data derived from 240,067 quarter milk samples from 93,529 dairy cows with mastitis; 238,235 cows from the same herds served as the control group. No target pathogen DNA was detected in 12% of the samples. In 49% of the positive samples, only one target species and in 19%, two species with one dominant species were present. The most common species in the samples with a single species only were coagulase-negative staphylococci (CNS) (43%), followed by Staphylococcus aureus (21%), Streptococcus uberis (9%), Streptococcus dysgalactiae (8%), Corynebacterium bovis (7%), and Escherichia coli (5%). On average, 36% of the study cows and 6% of the control cows had recorded mastitis treatments during lactation. The corresponding proportions were 16 and 6% at drying-off. For more than 75% of the treatments during lactation, diagnosis was acute clinical mastitis. In the milk samples from cows with a recorded mastitis treatment during lactation, CNS and S. aureus were most common, followed by streptococci. Altogether, 48% of the cows were culled during the study. Mastitis was reported as the most common reason to cull; 49% of study cows and 18% of control cows were culled because of mastitis. Culling was most likely if S. aureus was detected in the milk sample submitted during the culling year. Conclusions: The PCR test has proven to be an applicable method also for large-scale use in bacterial diagnostics. In the present study, microbiological diagnosis was unequivocal in the great majority of samples where a single species or two species with one dominating were detected. Coagulase-negative staphylococci and S. aureus were the most common species. S. aureus was also the most common pathogen among the culled cows, which emphasizes the importance of preventive measures.Peer reviewe

Prevalence of contagious and environmental mastitis-causing bacteria in bulk tank milk and its relationships with milking practices of dairy cattle herds in São Miguel Island (Azores)

This study aimed to assess the degree of contamination of bulk tank milk (BTM) by Staphylococcus spp. and coliform bacteria and to identify major milking practices that help perpetuate them in dairy cattle herds in São Miguel Island. In July 2014, BTM was sampled and a survey concerning local milking practices was conducted on 100 herds. Semi quantitative multiplex polymerase chain reaction detected coagulase-negative staphylococci, Escherichia coli, Staphylococcus aureus, and other coliform bacteria (Klebsiella oxytoca, Klebsiella pneumoniae, andSerratia marcescens) in 100, 75, 59, and 35 % of BTM, respectively. According to multivariable univariate models, on herds not using hot water for cleaning the milking machine and teat liners, there was at least 3.4 more odds (P<0.01) to have S. aureus or coliform bacteria contamination in BTM. The likelihoodoffinding S.aureus inBTMwas higher(P<0.001)on herds without high hygiene during milking, when milking mastitic cows at the end, on abrupt cessation of milking at dry-off, and official milk control implementation. The glove use also favored (odds ratio (OR) 5.8; P<0.01)thedetection ofcoliformbacteriainBTM.Poormilkingpracticesidentified in this study should be avoided in order to decrease S. aureus and coliform bacteria contamination of BTM. Other factors associated with milk quality in São Miguel Island also should be further investigated

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe