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Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens
Abstract
Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time-consuming. Multiplex real-time polymerase chain reaction (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity (Sp) of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR.
... milk samples taken at the monthly testmilking (non-aseptically collected). The PCR technique has shown high Se and specificity (Sp) in detecting bacteria in milk, compared to conventional culturing (Gillespie and Oliver, 2005;Koskinen et al., 2009;Paradis et al., 2012;Mahmmod et al., 2013c). Especially in samples with low bacterial concentration, PCR analysis seems better than BC in detecting bacteria (Taponen et al., 2009;Koskinen et al., 2010). ...
... Another disadvantage with PCR analysis is that it is not possible to save bacterial isolates for further testing, as culturing is not needed for PCR analysis. The PCR technology is applied routinely for analysis of milk samples in countries like Canada (Can West DHI, 2016), Denmark (SEGES, 2012) and Finland (Hiitio, 2016), but few studies have been published reporting the accuracy of this test for more than one mastitis pathogen, or under field conditions (Koskinen et al., 2010;Paradis et al., 2012). Therefore, the objective of this study was to estimate and compare the diagnostic accuracy of PCR analysis of composite milk samples routinely collected in the field using non-aseptic sampling technique and conventional BC of quarter milk samples collected aseptically, when used to identify cows with IMI with four common mastitis pathogens. ...
... Sampled cows were divided into two populations, one with a somatic cell count (SCC) at the day of test milking of <100,000 cells/ml and one with SCC ≥100,000 cells/ml at the day of test milking. LCA analysis was used for a similar purpose (Cederlof et al., 2012;Paradis et al., 2012;Mahmmod et al., 2013b), although the exact estimates of Se and Sp differ somewhat. The Se for both PCR analysis and BC were higher in the studies of Cederlof et al. (2012) and Paradis et al. (2012), while the Se for PCR analysis was higher and the Se of BC was lower in the study by Mahmmod et al. (2013b). ...
Real-time PCR analysis of milk samples is a fast method to identify intramammary infections (IMI) in
dairy cows, and has the potential to be used for routine analysis of test milking composite milk samples.
However, the results of the PCR analysis can be difficult to interpret. The objective of this study was to
compare the sensitivity (Se) and specificity (Sp) of PCR analysis of composite milk samples, and conventional bacteriological culturing (BC) of quarter milk samples, when used to identify cows with IMI.
The comparisons were performed for IMI with four common udder pathogens; Staphylococcus aureus
(S aureus), Streptococcus dysgalactiae (Str dysgalactiae), Str uberis and coagulase negative staphylococci
(CoNS). The Se and Sp of real-time PCR (SePCR; SpPCR) and BC (SeBC; SpBC) was simultaneously estimated
using latent class analysis (LCA), studying one pathogen at the time. Milk samples from 970 dairy cows
from 25 herds were included. Aseptically collected quarter milk samples taken at the day before test
milking (TM), at the day of TM, and at the day after TM, were analyzed using BC. Non-aseptically collected composite milk samples taken at the day of TM were analyzed using PCR. Moreover, the composite
milk somatic cell count (SCC) was recorded and summarized by diagnostic test and bacterial finding. LCA
was first performed using only test results from samples taken at the day of TM, but in a second analysis
BC results from the three consecutive samplings, interpreted in parallel, were included. The SePCR was
significantly higher than the SeBC for S aureus, Str dysgalactiae and CoNS in the first analysis, but only for
CoNS in the second analysis. The SpPCR was significantly lower than the SpBC for Str dysgalactiae and CoNS.
In conclusion, using PCR analysis of composite milk samples, as a diagnostic tool for identifying cows with
IMI increased the Se for all the pathogens investigated (although not always significantly), while Sp in
general remained on a similar level, compared to BC of quarter milk samples. The use of repeated quarter
milk sampling improved the SeBC, making the results of PCR analysis and BC more similar. However, the
SCC of cows with IMI according to BC was higher than for cows with IMI according to PCR, suggesting that
some of the cows with IMI according to PCR did not have an active inflammation. Hence, extra caution is
needed when decisions about treatment of IMI are based on PCR
... Estimates of Se for BC and qPCR for S. agalactiae in quarter milk samples using LCA have also been published by Paradis et al. (2012), using priors for BC from Dinsmore et al. (1991) and qPCR priors from Gillespie and Oliver (2005). However, due to a lack of BC positive cases and a very low number of qPCR positive cases, the estimates obtained in this study were very close to the prior estimates (Paradis et al., 2012). ...
... Estimates of Se for BC and qPCR for S. agalactiae in quarter milk samples using LCA have also been published by Paradis et al. (2012), using priors for BC from Dinsmore et al. (1991) and qPCR priors from Gillespie and Oliver (2005). However, due to a lack of BC positive cases and a very low number of qPCR positive cases, the estimates obtained in this study were very close to the prior estimates (Paradis et al., 2012). The applied priors were estimated based on BC being a perfect reference test. ...
... Therefore, during the last decade, several molecular techniques such as PCR and next-generation sequencing have been introduced to enlarge the spectrum of methods for mastitis diagnostics. These new approaches provide better sensitivity in detecting bacteria and especially bacterial DNA in milk in comparison to CC [12,[14][15][16]. Due to the increase in knowledge and the availability of modern analysis techniques, new perspectives have emerged regarding the colonization of microbial communities in various organs and regions of the body [17]. ...
Bovine udder health is an important factor for animal wellbeing and the dairy farm economy. Thus, researchers aim to understand factors causing mastitis. The gold standard for diagnosing mastitis in cows is the conventional culturing of milk samples. However, during the last few years, the use of molecular methods has increased. These methods, especially sequencing, provide a deeper insight into the diversity of the bacterial community. Yet, inconsistent results regarding the mammary microbiome have been published. This study aimed to evaluate the udder health of eight dairy cows at seven days postpartum with the standard methods in veterinary practice. Additionally, swabs from the teat canal and milk samples were analyzed using 16S rRNA gene amplicon sequencing. The sensitive low-biomass milk samples displayed only a few contaminations even though they were sampled in a field environment. In healthy udders, no bacterial communities were detected by the bacterial culture nor the 16S rRNA gene amplicons. The results from the standard examination of the cows, the cell count, and the bacteriological examination were comparable with the results from 16S rRNA gene amplicon sequencing when cows displayed subclinical or latent mastitis. Besides the pathogen detected in bacterial culturing, a second bacterial strain with low but significant abundance was detected by sequencing, which might aid in the understanding of mastitis incidence. In general, molecular biological approaches might lead to promising insights into pathological events in the udder and might help to understand the pathomechanism and infection source via epidemiological analyses.
... Milk samples were analyzed using qPCR through a commercial diagnostic kit (Mastitis 4E kit; DNA Diagnostic A/S) according to the manufacturer's instructions. This technique showed sensitivity and specificity, respectively, of ≥0.95 and ≥0.99 for the contagious pathogens [30][31][32]. This kit allows bacterial DNA extraction, identification, and quantification of S. aureus, Str. ...
Mastitis is one of the major diseases of dairy cows that affects milk quality and quantity and increases the potential risk for the presence of antimicrobial residues (AR) in milk, which could lead to the development of antimicrobial resistance (AMR) among human pathogens. Even if the presence of AR in milk and milk products is low in many countries, the threat is not negligible and cannot be ignored. These problems may be investigated by applying a One Health approach, and this prevalence study aimed to estimate the risks for human health related to milk production applied to dairy herds in Lombardy. Three hundred thirty-one bulk tank milk samples were randomly collected and analyzed by CombiFoss 7 and MilkoScan 7 (milk quality, bacteria, and somatic cell count), an HPLC system coupled to a Q-Exactive Orbitrap (AR), and qPCR (contagious pathogens). The data were analyzed by a generalized linear model. The results showed a relatively high prevalence of contagious pathogens (S. aureus 28.1%; Str. agalactiae 7.3%; M. bovis 3%), which primarily affect milk nutritional components decreasing mainly milk fat content (range 1%–2.5%), but did not show them to be associated to an increase of the risk of antimicrobial residues. These latter ones were recovered only in 7/331 samples at concentrations far below official MLRs. The results support currently active surveillance programs’ efficacy in reducing AR risks, which may be further improved by prioritizing them based on geographical area characteristics.
... To determine the impact of the prior on the posterior densities, two additional models were ran using weakly informative priors on the prevalence or for the tests Se and Sp (Depaoli et al. 2020;Natesan Batley and Hedges 2021;Paradis et al. 2012). In model 2, we used relaxed estimates for the prevalence in the two populations. ...
The sensitivity (Se) and specificity (Sp) of three diagnostic tests for the detection of Campylobacter fetus venerealis (Cfv) using field samples were estimated using a Bayesian latent class model (BLCM), accounting for the absence of a gold standard. The tests included in this study were direct immunofluorescence antibody test (IFAT), polymerase chain reaction (PCR), and real-time PCR (RT-PCR). Twelve farms from two different populations were selected and bull prepuce samples were collected. The IFAT was performed according to the OIE Manual. The conventional PCR was performed as multiplex, targeting the gene nahE for C. fetus species identification and insertion element ISCfe1 for Cfv identification. The RT-PCR was performed as uniplex: one targeting the gene nahE for C. fetus and the other targeting the insertion ISCfe1 (ISC2) for Cfv. Results from the BLCM showed a median Se of 11.7% (Bayesian credibility interval (BCI): 1.93–29.79%), 53.7% (BCI: 23.1–95.0%), and 36.1% (BCI: 14.5–71.7%) for IFAT, PCR, and RT-PCR respectively. The Sp were 94.5% (BCI: 90.1–97.9%), 97.0% (BCI: 92.9–99.3%), and 98.4% (BCI: 95.3–99.7%) for IFAT, PCR, and RT-PCR respectively. The correlation between PCR and RT-PCR was positive and low in samples from both sampled population (0.63% vs 8.47%). These results suggest that diagnostic sensitivity of the studied tests is lower using field samples than using pure Cfv strains.
... The transformation makes it possible to fit the resulting data to generalized linear mixed models to account for Poisson-distributed fluctuations when the number of molecule count is low. Similar Bayesian approaches for analyzing qPCR data have been used in several other reports [47][48][49][50][51]. While Actin was designated as an ICG, it was found to be differentially expressed following heat stress and, therefore, no longer utilized as an internal control here and the model was run in the "naive" form (i.e., no control gene). ...
The ability of corals to withstand changes in their surroundings is a critical survival mechanism for coping with environmental stress. While many studies have examined responses of the coral holobiont to stressful conditions, its capacity to reverse responses and recover when the stressor is removed is not well-understood. In this study, we investigated among-colony responses of Pocillopora acuta from two sites with differing distance to the mainland (Kusu (closer to the mainland) and Raffles Lighthouse (further from the mainland)) to heat stress through differential expression analysis of target genes and quantification of photophysiological metrics. We then examined how these attributes were regulated after the stressor was removed to assess the recovery potential of P. acuta. The fragments that were subjected to heat stress (2 °C above ambient levels) generally exhibited significant reduction in their endosymbiont densities, but the extent of recovery following stress removal varied depending on natal site and colony. There were minimal changes in chl a concentration and maximum quantum yield (Fv/Fm, the proportion of variable fluorescence (Fv) to maximum fluorescence (Fm)) in heat-stressed corals, suggesting that the algal endosymbionts’ Photosystem II was not severely compromised. Significant changes in gene expression levels of selected genes of interest (GOI) were observed following heat exposure and stress removal among sites and colonies, including Actin, calcium/calmodulin-dependent protein kinase type IV (Camk4), kinesin-like protein (KIF9), and small heat shock protein 16.1 (Hsp16.1). The most responsive GOIs were Actin, a major component of the cytoskeleton, and the adaptive immune-related Camk4 which both showed significant reduction following heat exposure and subsequent upregulation during the recovery phase. Our findings clearly demonstrate specific responses of P. acuta in both photophysiological attributes and gene expression levels, suggesting differential capacity of P. acuta corals to tolerate heat stress depending on the colony, so that certain colonies may be more resilient than others.
... Bulk tank milk samples were analysed using qPCR. This technique showed to have a sensitivity of Se and Sp of qPCR respectively !0.95 and !0.99 for the contagious pathogens (Paradis et al. 2012;Hiiti€ o et al. 2016;Timonen et al. 2017). ...
... Bulk tank milk samples were analysed using qPCR. This technique showed to have a sensitivity of Se and Sp of qPCR respectively !0.95 and !0.99 for the contagious pathogens (Paradis et al. 2012;Hiiti€ o et al. 2016;Timonen et al. 2017). ...
Data on the prevalence of major contagious pathogens in bulk tank milk (BTM) in Italy are generally not available. The availability of Real-Time PCR procedures (qPCR) to perform BTM analysis by represents an important step to define herd health status. Therefore, a cross-sectional epidemiological study was designed to assess the prevalence of contagious pathogens and Prototheca spp in BTM samples. The study was performed on 581 herds from four districts in the west Lombardy region of Italy. Additionally, the relationship between pathogens in BTM and SCC or milk yield; the presence of an association between four risk factors (district, herd size, average milk yield and SCC) with pathogens in BTM were assessed. The overall data showed that S. aureus was recovered in 42% of the herds, Str. agalactiae in 10%, Prototheca spp in 11% and M. bovis in 1.5% of the herds. The GLM model applied showed a significant influence of BTM results, district, herd size and their interactions on SCC and on milk yield variance. Particularly, S. aureus or Str. agalactiae have a significant effect on milk yield variability and, in a lesser extent, on SCC. The very high prevalence of contagious pathogens significantly affects milk characteristics and yield, thus affecting economic sustainability of the herds, and suggests the need to implement control programmes to decrease the prevalence of contagious pathogens, This will also allow to decrease the use of antimicrobials and to improve cow welfare.
• Highlights
• First study on a large sample of Italian dairy herds on the prevalence of contagious pathogens in bulk tank milk samples. The prevalence value observed exceeded 50%.
• First study estimating the prevalence of M. bovis in bulk tank milk in a large sample of Italian dairy herds, and the prevalence observed was 1.5%.
• Prevalence of contagious pathogens has a significant influence on milk yield and SCC.
• Bulk tank milk SCC confirmed to have a low accuracy to identify infected herds.
... There is a need of a perfect gold standard test for detection of bovine mastitis pathogens, which is rapid and accurate, specific, and sensitive and can be easily performed. Various studies focused on the development of a multiplex real-time PCR assay for detection of major mastitis pathogens, and this is showing a high potential and future prospects due to high sensitivity and specificity (Gillespie and Oliver 2005;Paradis et al. 2012). ...
Accurate diagnosis of disease is the major step between the cause and cure of disease. An economical, reliable, and rapid diagnostic tool is fundamental for the management of udder health. The earlier the disease is identified, the less will be the damage; keeping this in mind, many efforts are being made to develop reliable diagnostic tools for use on farm. However, traditional gold standard methods including somatic cell count and microbial culturing are still in use. They are partially being replaced with polymerase chain reaction and sequencing-based tests. Nanotechnology and protein-based tests have also gained lot of attention and some of them are potential candidate of future diagnostic tests for bovine mastitis. Research laboratories are struggling to develop simple, economical, and user-friendly biosensor-based methods that can be performed on farm for rapid diagnosis. The combination of both genomic and proteomic approaches, together with further involvement of nanotheranostic technologies and other sensors, will assist in the quest of better mastitis diagnostic tools.
... In the absence of a gold/reference standard, latent class models implemented through Bayesian estimation procedures have been successfully used to estimate true disease prevalence and diagnostic performance for two or more diagnostic tests applied to samples from test subjects of unknown underlying disease status (Berkvens et al., 2006;Fablet et al., 2010;Haley et al., 2011;Paradis et al., 2012;Paul et al., 2013). Published standards for reporting diagnostic accuracy using Bayesian Latent Class Models were followed (Kostoulas et al., 2017, Appendix A in supplementary file). ...
Cattle are a reservoir for Escherichia coli O157 and they shed the pathogen in their feces. Fecal contaminants on the hides can be transferred onto carcasses during processing at slaughter plants, thereby serving as a source of foodborne infection in humans. The detection of E. coli O157 in cattle feces is based on culture, immunological, and molecular methods We evaluated the diagnostic sensitivity and specificity of one culture- and two PCR-based tests for the detection of E. coli O157 in cattle feces, and its true prevalence using a Bayesian implementation of latent class models. A total of 576 fecal samples were collected from the floor of pens of finishing feedlot cattle in the central United States during summer 2013. Samples were enriched and subjected to detection of E. coli O157 by culture (immunomagnetic separation, plating on a selective medium, latex agglutination, and indole testing), conventional PCR (cPCR), and multiplex quantitative PCR (mqPCR). The statistical models assumed conditional dependence of the PCR tests and high specificity for culture (mode = 99%; 5th percentile = 97%). Prior estimates of test parameters were elicited from three experts. Estimated posterior sensitivity (posterior median and 95% highest posterior density intervals) of culture, cPCR, and mqPCR was 49.1% (44.8–53.4%), 59.7% (55.3–63.9%), and 97.3% (95.1–99.0%), respectively. Estimated posterior specificity of culture, cPCR, and mqPCR were 98.7% (96.8–99.8%), 94.1% (87.4–99.1%), and 94.8% (84.1–99.9%), respectively. True prevalence was estimated at 91.3% (88.1–94.2%). There was evidence of a weak conditional dependence between cPCR and mqPCR amongst test positive samples, but no evidence of conditional dependence amongst test negative samples. Sensitivity analyses showed that overall our posterior inference was rather robust to the choice of priors, except for inference on specificity of mqPCR, which was estimated with considerable uncertainty. Our study evaluates performance of three diagnostic tests for detection of E. coli O157 in feces of feedlot cattle which is important for quantifying true fecal prevalence and adjusting for test error in risk modeling.
... Most studies that evaluated test characteristics of mastitis PCR systems used BC of quarter milk samples as the reference method, although some authors stated that its sensitivity and specificity is limited (Krömker et al., 2008;Paradis et al., 2012). Studies in which quarter milk samples or composite samples were analyzed in parallel with both test methods detected more pathogens by qPCR than by BC (Taponen et al., 2009;Koskinen et al., 2010;Spittel and Hoedemaker, 2012). ...
The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis. In addition, PCR was performed on pooled herd milk samples containing milk aliquots from all lactating cows in each of the 51 herds. Only 1 out of the 51 herds was found PCR positive for Streptococcus agalactiae in bulk tank and pooled herd milk samples, and cultured quarter milk samples. Spearman's rank correlations between the cycle threshold value of bulk tank milk PCR and the apparent within-herd prevalence were calculated in regard to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis. For these pathogens, significant correlations were found. If 1 bulk tank milk sample per herd was tested, the estimated within-herd prevalence thresholds for 90% probability of detection were 27.6% for Staphylococcus aureus, 9.2% for Streptococcus dysgalactiae, and 13.8% for Streptococcus uberis on the cow level. On the quarter level, the within-herd prevalence had to be at least 32.6% for Staphylococcus aureus, 1.7% for Streptococcus dysgalactiae, and 4.3% for Streptococcus uberis to detect a herd as positive using a single bulk milk sample. The results indicate that mastitis pathogens in bulk tank milk can be identified by the applied PCR assay. Bulk tank milk examination is not a reliable tool for the identification of the named pathogens by single testing, but might be a valuable monitoring tool when used frequently with repeated testing. Furthermore, this approach could be a useful monitoring tool for detecting new pathogen occurrence in the herd.
... The diagnostic sensitivity of the test was 96.15% and specificity was 98.60% [39]. Paradis et al. [40] used an LCA model for estimation of sensitivity and specificity of multiplex real-time PCR assay for the detection of S. aureus, S. uberis, E. coli, and S. agalactiae. Sensitivity was found to be in the range of 66%e96% and specificity was !99% for all the target species. ...
For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. Detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10⁴ CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies.
... Latent class analysis provides an alternative method for evaluating diagnostic tests when no perfect reference test is available (Dohoo et al., 2009). Although it has been used in PCR validation studies (Cederlöf et al., 2012;Paradis et al., 2012), latent class analysis was not appropriate here because the test sensitivity and specificity were not constant across subpopulations, therefore failing to meet one criterion required for this analysis (Enøe et al., 2000). ...
The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of Streptococcus uberis and Staphylococcus aureus in bovine milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical mastitis cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy cows, during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. aureus infection. Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85–89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early lactation, specifically for diagnosing Strep. uberis. Thus, PCR should not be used as the only tool for diagnosing mastitis in early lactation.
... Analysis of isolates and milk samples utilizing molecular methods such as PCR are described in the literature (Phuektes et al., 2001;Meiri-Bendek et al., 2002;McDonald et al., 2005;Paradis et al., 2012), and PCR testing for S. agalactiae is now widely commercially available. The PathoProof Mastitis PCR Assay (Thermo Fisher Scientific, Vintaa, Finland) is a commercially available multiplex real-time test kit that simultaneously detects the presence of several different bacterial species in milk (Koskinen et al., 2010), one of which is S. agalactiae, and which is purported to have the advantage over microbiological culture in that it is more sensitive and specific, particularly with its ability to identify bacteria that are no longer viable or whose growth is inhibited (Taponen et al., 2009;Koskinen et al., 2010;Mweu et al., 2012b). ...
Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64°C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen.
... They used the same Ct cut-off value of 37, but the statistical method of analysing the results differed (Latent Class Analysis). Paradis et al. (2012) used a different multiplex real-time PCR assay, resulting in a median Se of 72 to 79%. In our calculations, a positive result for Staph. ...
Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85·7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83·0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97·0 and 95·8% compared with BC. For Staphylococcus spp., the corresponding figures were 86·7 and 75·4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37·0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.
... Relatively few published studies have evaluated the accuracy of different PCR techniques based on comparison to imperfect reference tests (Ahmadi et al., 2010;Friendship et al., 2010). On quarter-level, Paradis et al. (2012) estimated the diagnostic accuracy of a multiplex real-time PCR and BC for subclinical IMI with S. aureus using Bayesian analysis. The authors estimated the Se of BC of 66% and 83% based on the IMI definition of 10 cfu and 1 cfu per 0.01 ml, while the Se of PCR was 95% and 88%, respectively. ...
Danish farmers can order a real-time PCR mastitis diagnostic test on routinely taken cow-level samples from milk recordings. Validation of its performance in comparison to conventional mastitis diagnostics under field conditions is essential for efficient control of intramammary infections (IMI) with Staphylococcus aureus (S. aureus). Therefore, the objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR, bacterial culture (BC) and California mastitis test (CMT) for the diagnosis of the naturally occurring IMI with S. aureus in routinely collected milk samples using latent class analysis (LCA) to avoid the assumption of a perfect reference test. Using systematic random sampling, a total of 609 lactating dairy cows were selected from 6 dairy herds with bulk tank milk PCR cycle threshold (Ct) value ≤39 for S. aureus. At routine milk recordings, automatically obtained cow-level (composite) milk samples were analyzed by PCR and at the same milking, 2436 quarter milk samples were collected aseptically for BC and CMT. Results showed that 140 cows (23%) were positive for S. aureus IMI by BC while 170 cows (28%) were positive by PCR. Estimates of Se and Sp for PCR were higher than test estimates of BC and CMT. SeCMT was higher than SeBC however, SpBC was higher than SpCMT. SePCR was 91%, while SeBC was 53%, and SeCMT was 61%. SpPCR was 99%, while SpBC was 89%, and SpCMT was 65%. In conclusion, PCR has a higher performance than the conventional diagnostic tests (BC and CMT) suggesting its usefulness as a routine test for accurate diagnosis of S. aureus IMI from dairy cows at routine milk recordings. The use of LCA provided estimates of the test characteristics for two currently diagnostic tests (BC, CMT) and a novel technique (real-time PCR) for diagnosing S. aureus IMI under field conditions at routine milk recordings in Denmark.
Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.
Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n = 67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.
Criteria for diagnosing intramammary infections (IMI) have been debated for many years. Factors that may be considered in making a diagnosis include the organism of interest being found on culture, the number of colonies isolated, whether or not the organism was recovered in pure or mixed culture, and whether or not concurrent evidence of inflammation existed (often measured by somatic cell count). However, research using these criteria has been hampered by the lack of a "gold standard" test (i.e., a perfect test against which the criteria can be evaluated) and the need for very large data sets of culture results to have sufficient numbers of quarters with infections with a variety of organisms. This manuscript used 2 large data sets of culture results to evaluate several definitions (sets of criteria) for classifying a quarter as having, or not having an IMI by comparing the results from a single culture to a gold standard diagnosis based on a set of 3 milk samples. The first consisted of 38,376 milk samples from which 25,886 triplicate sets of milk samples taken 1 wk apart were extracted. The second consisted of 784 quarters that were classified as infected or not based on a set of 3 milk samples collected at 2-d intervals. From these quarters, a total of 3,136 additional samples were evaluated. A total of 12 definitions (named A to L) based on combinations of the number of colonies isolated, whether or not the organism was recovered in pure or mixed culture, and the somatic cell count were evaluated for each organism (or group of organisms) with sufficient data. The sensitivity (ability of a definition to detect IMI) and the specificity (Sp; ability of a definition to correctly classify noninfected quarters) were both computed. For all species, except Staphylococcus aureus, the sensitivity of all definitions was <90% (and in many cases<50%). Consequently, if identifying as many existing infections as possible is important, then the criteria for considering a quarter positive should be a single colony (from a 0.01-mL milk sample) isolated (definition A). With the exception of "any organism" and coagulase-negative staphylococci, all Sp estimates were over 94% in the daily data and over 97% in the weekly data, suggesting that for most species, definition A may be acceptable. For coagulase-negative staphylococci, definitions B (2 colonies from a 0.01-mL milk sample) raised the Sp to 92 and 95% in the daily and weekly data, respectively. For "any organism," using definition B raised the Sp to 88 and 93% in the 2 data sets, respectively. The final choice of definition will depend on the objectives of study or control program for which the sample was collected.
The primary purpose of this study was to develop a set of criteria to serve as a pseudo-gold standard for what constitutes an intramammary infection using data from 3 consecutive quarter milk samples taken 1 wk apart. Data from lactating cows in 90 dairy herds in 4 Canadian provinces were used to generate the data sets (profiles) used in the conjoint analysis to elicit expert opinions on the topic. The experts were selected from the participants (n=23) in the 2007 Mastitis Research Workers' Conference in Minneapolis and from a series of mastitis laboratory courses for bovine practitioners (n=25) in the Netherlands. Three-week udder quarter profiles with specific combinations of somatic cell count, bacterial species isolated, and plate colony count were selected and included in the conjoint analysis based on the desire to achieve even distributions in the categories of 6 constructed variables. The participants were presented with 3 sets of cards with 20 cards in each set. On each card, they were asked to assign a probability of infection on the middle day (test day) in the 3-wk profile. Depending on the set of cards, they were asked only to be concerned with the probability of infection with coagulase-negative staphylococci, Escherichia coli, or Staphylococcus aureus. These 3 organisms were chosen to represent a minor pathogen, a major environmental pathogen, and a major contagious pathogen, respectively. The assigned probabilities for each organism were cross-tabulated according to the number of times the organism of interest was isolated in the 3-wk period, how many colonies of the organism of interest were isolated on the test day, and the somatic cell count (<or= or >200,000 cells/mL). There was considerable variation in the assigned probabilities within each of the combinations of factors. The median, minimum, and maximum values of the assigned probabilities for each combination were computed. The combinations with a median probability >50% were considered intramammary infection-positive and included as a criterion in the consensus standard. This yielded 4 possible criteria, which were condensed to the following 2 by consensus at the 2008 Mastitis Research Workers' Conference in Toronto: 1) the organism of interest was isolated on the test day with at least 10 colonies (1,000 cfu/mL), and 2) the organism of interest was isolated at least twice in the 3-wk period.
In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The "no-growth" samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10(3) to 10(7) bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-beta-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.
Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical specimens, including specimens from patients with ongoing antimicrobial therapy.
Streptococcus agalactiae continues to be a major cause of subclinical mastitis in dairy cattle and a source of economic loss for the industry. Veterinarians are often asked to provide information on herd level control and eradication of S. agalactiae mastitis. This review collects and collates relevant publications on the subject. The literature search was conducted in 1993 on the Agricola database. Articles related to S. agalactiae epidemiology, pathogen identification techniques, milk quality consequences, and control, prevention, and therapy were included. Streptococcus agalactiae is an oblique parasite of the bovine mammary gland and is susceptible to treatment with a variety of antibiotics. Despite this fact, where state or provincial census data are available, herd prevalence levels range from 11% (Alberta, 1991) to 47% (Vermont, 1985). Infection with S. agalactiae is associated with elevated somatic cell count and total bacteria count and a decrease in the quantity and quality of milk products produced. Bulk tank milk culture has, using traditional milk culture techniques, had a low sensitivity for identifying S. agalactiae at the herd level. New culture methods, using selective media and large inocula, have substantially improved the sensitivity of bulk tank culture. Efficacy of therapy on individual cows remains high. Protocols for therapy of all infected animals in a herd are generally successful in eradicating the pathogen from the herd, especially if they are followed up with good udder hygiene techniques.
To improve diagnosis of mastitis in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of the four major bacterial causes of bovine mastitis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. The target sequence was the 16S to 23S rRNA spacer regions. The performance of the assay was examined with 117 milk samples collected from a subclinically infected herd, and the diagnostic specificities and sensitivities of the multiplex PCR were compared with conventional culture. PCR was significantly more sensitive than culture for detection of S. aureus and S. uberis, but there were no significant differences in sensitivities between PCR and culture for the detection of S. agalactiae and S. dysgalactiae. The results suggest that this multiplex PCR assay could be used as an alternative method in routine diagnosis for rapid, sensitive, and specific simultaneous detection of S. aureus, S. agalactiae, S. dysgalactiae, and S. uberis in milk samples.
The objective of this study was to examine the characteristics of milk samples submitted for microbiological examination at the Wisconsin Veterinary Diagnostic Laboratory between 1994 and 2001. Results (n = 83,650) of microbiological testing of milk samples (n = 77,172) submitted to the Wisconsin Veterinary Diagnostic Laboratory from January 1994 until June 2001 were analyzed. Submissions included milk samples obtained from cases of clinical and subclinical mastitis as well as samples obtained for mastitis surveillance programs. Results were recorded as no growth, contaminated, or identified as specific bacterial pathogens. Statistical analysis was performed to determine trends in the isolation of mastitis pathogens. The proportion of samples identified as contaminated decreased from 20.6 (1997) to 9.5% (2001). The proportion of samples coded as no growth increased from 22.6 (1994) to 49.7% (2001). Isolation of Staphylococcus aureus decreased from 17.7% (1994) of isolates to 9.7% (2001), while isolation of Streptococcus agalactiae decreased from 8.1 (1994) to 3.0% (2001). Coagulase-negative Staphylococcus spp. were isolated from 12.7 to 17.5%, environmental Streptococcus spp. were isolated from 11.6 to 20.1%, and Escherichia coli were isolated from 3.1 to 6.7% of all isolates. No growth and contaminated samples comprised almost 50% of total submissions, and it is important that producers have proper expectations when submitting milk samples. The proportion of isolates identified as Staph. aureus and Strep. agalactiae decreased, suggesting the proportion of contagious bacteria causing mastitis has decreased. Environmental and contagious pathogens demonstrated characteristic differences by season.
In this chapter, we describe the use of R to summarize Bayesian models with several unknown parameters. In learning about parameters of a normal population or multinomial parameters, posterior inference is accomplished by simulating from distributions of standard forms.
R is a rich environment for statistical computing and has many capabilities for exploring data in its base package. In addition, R contains a collection of functions for simulating and summarizing the familiar one-parameter probability distributions. One goal of this chapter is to provide a brief introduction to basic commands for summarizing and graphing data. We illustrate these commands on a dataset about students in an introductory statistics class. A second goal of this chapter is to introduce the use of R as an environment for programming Monte Carlo simulation studies. We describe a simple Monte Carlo study to explore the behavior of the two-sample t statistic when testing from populations that deviate from the usual assumptions. We will find these data analysis and simulation commands very helpful in Bayesian computation.
In the previous two chapters, two types of strategies were used in the summarization of posterior distributions. If the sampling density has a familiar functional form, such as a member of an exponential family, and a conjugate prior is chosen for the parameter, then the posterior distribution often is expressible in terms of familiar probability distributions.
This random number generator was OK when it was written, but is not adequate for modern large-scale simulations.
Many analyses of results from multiple diagnostic tests assume the tests are statistically independent conditional on the true disease status of the subject. This assumption may be violated in practice, especially in situations where none of the tests is a perfectly accurate gold standard. Classical inference for models accounting for the conditional dependence between tests requires that results from at least four different tests be used in order to obtain an identifiable solution, but it is not always feasible to have results from this many tests. We use a Bayesian approach to draw inferences about the disease prevalence and test properties while adjusting for the possibility of conditional dependence between tests, particularly when we have only two tests. We propose both fixed and random effects models. Since with fewer than four tests the problem is nonidentifiable, the posterior distributions are strongly dependent on the prior information about the test properties and the disease prevalence, even with large sample sizes. If the degree of correlation between the tests is known a priori with high precision, then our methods adjust for the dependence between the tests. Otherwise, our methods provide adjusted inferences that incorporate all of the uncertainty inherent in the problem, typically resulting in wider interval estimates. We illustrate our methods using data from a study on the prevalence of Strongyloides infection among Cambodian refugees to Canada.
Bovine mastitis, the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. Traditionally, methods of detection have included estimation of somatic cell counts, an indication of inflammation, measurement of biomarkers associated with the onset of the disease (e.g. the enzymes N-acetyl-β-D-glucosaminidase and lactate dehydrogenase) and identification of the causative microorganisms, which often involves culturing methods. These methods have their limitations and there is a need for new rapid, sensitive and reliable assays. Recently, significant advances in the identification of nucleic acid markers and other novel biomarkers and the development of sensor-based platforms have taken place. These novel strategies have shown promise, and their advantages over the conventional tests are discussed.
Costs and feasibility of extensive sample collection and processing are major obstacles to mastitis epidemiology research. Studies are often consequentially limited, and fundamental mastitis researchers rarely have the opportunity to conduct their work in epidemiologically valid populations. To mitigate these limitations, the Canadian Bovine Mastitis Research Network has optimized research funds by creating a data collection platform to provide epidemiologically meaningful data for several simultaneous research endeavors. This platform consists of a National Cohort of Dairy Farms (NCDF), Mastitis Laboratory Network, and Mastitis Pathogen Culture Collection. This paper describes the implementation and operation of the NCDF, explains its sampling protocols and data collection, and documents characteristics, strengths and limitations of these data for current and potential users. The NCDF comprises 91 commercial dairy farms in 6 provinces sampled over a 2-yr period. Primarily Holstein-Friesian herds participating in Dairy Herd Improvement milk recording were selected in order to achieve a uniform distribution among 3 strata of bulk tank somatic cell counts and to reflect regional proportions of freestall housing systems. Standardized protocols were implemented for repeated milk samplings on clinical mastitis cases, fresh and randomly selected lactating cows, and cows at dry-off and after calving. Just fewer than 133,000 milk samples were collected. Demographic and production data were recorded at individual cow and farm levels. Health management data are documented and extensive questionnaire data detailing farm management and cleanliness information are also captured. The Laboratory Network represents coordinated regional mastitis bacteriology laboratories using standardized procedures. The Culture Collection archives isolates recovered from intramammary infections of cows in the NCDF and contains over 16,500 isolates, all epidemiologically cross-referenced between linked databases. The NCDF is similar to Canadian dairies in relation to mean herd size, average production, and freestall percentages. Pathogen recovery was greater than anticipated, particularly for coagulase-negative staphylococci and Corynebacterium spp. International scientists are encouraged to use this extensive archive of data and material to enhance their own mastitis research.
The Gibbs sampler, the algorithm of Metropolis and similar iterative simulation methods are potentially very helpful for summarizing multivariate distributions. Used naively, however, iterative simulation can give misleading answers. Our methods are simple and generally applicable to the output of any iterative simulation; they are designed for researchers primarily interested in the science underlying the data and models they are analyzing, rather than for researchers interested in the probability theory underlying the iterative simulations themselves. Our recommended strategy is to use several independent sequences, with starting points sampled from an overdispersed distribution. At each step of the iterative simulation, we obtain, for each univariate estimand of interest, a distributional estimate and an estimate of how much sharper the distributional estimate might become if the simulations were continued indefinitely. Because our focus is on applied inference for Bayesian posterior distributions in real problems, which often tend toward normality after transformations and marginalization, we derive our results as normal-theory approximations to exact Bayesian inference, conditional on the observed simulations. The methods are illustrated on a random-effects mixture model applied to experimental measurements of reaction times of normal and schizophrenic patients.
Bovine mastitis, the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. Traditionally, methods of detection have included estimation of somatic cell counts, an indication of inflammation, measurement of biomarkers associated with the onset of the disease (e.g. the enzymes N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase) and identification of the causative microorganisms, which often involves culturing methods. These methods have their limitations and there is a need for new rapid, sensitive and reliable assays. Recently, significant advances in the identification of nucleic acid markers and other novel biomarkers and the development of sensor-based platforms have taken place. These novel strategies have shown promise, and their advantages over the conventional tests are discussed.
The sensitivity, specificity, and positive and negative predictive values were compared for six methods of collecting or culturing milk samples for the diagnosis of chronic Streptococcus agalactiae intramammary infection. Cows in four dairy herds were cultured three times in 2 wk to determine infection status. At the second sample period, individual quarter and composite milk samples were taken before and immediately after milking, and two volumes of milk from the composite samples were streaked on culture plates. The sensitivity, specificity, and predictive values for these cultures ranged between 95 and 100% for all diagnostic methods, and no significant differences were found between methods. The results indicate that when either quarter or composite samples are collected before or immediately after milking, 95 to 100% of S. agalactiae culture-positive cows will be infected with S. agalactiae in herds with a prevalence of S. agalactiae between 35 and 55%. A similar proportion of culture-negative cows will be uninfected.
Twenty-one quarters of seven cows were experimentally infected with Staphylococcus aureus (ATCC 29740) to study the shedding pattern in quarter milk samples. Of 991 consecutive quarter milk samples collected from infected quarters during the trial, 745 were positive for S. aureus by bacteriological culture. The sensitivity of a single quarter milk sample to determine infection status of a quarter was 74.5% based on the mean of each gland's recovery pattern. Sensitivity of bacterial culture increased to 94% and 98% by including a second and a third consecutive sample. Because S. aureus is shed in a cyclical manner from mammary glands, consecutive samples would be advisable for accurate diagnosis of infected quarters.
It is common in population screening surveys or in the investigation of new diagnostic tests to have results from one or more tests investigating the same condition or disease, none of which can be considered a gold standard. For example, two methods often used in population-based surveys for estimating the prevalence of a parasitic or other infection are stool examinations and serologic testing. However, it is known that results from stool examinations generally underestimate the prevalence, while serology generally results in overestimation. Using a Bayesian approach, simultaneous inferences about the population prevalence and the sensitivity, specificity, and positive and negative predictive values of each diagnostic test are possible. The methods presented here can be applied to each test separately or to two or more tests combined. Marginal posterior densities of all parameters are estimated using the Gibbs sampler. The techniques are applied to the estimation of the prevalence of Strongyloides infection and to the investigation of the diagnostic test properties of stool examinations and serologic testing, using data from a survey of all Cambodian refugees who arrived in Montreal, Canada, during an 8-month period.
Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhib- itors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase ac- tivity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important im- plications for clinical and public health investigations, espe- cially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constitu- ents (e.g., organic and phenolic compounds, glycogen, fats, and Ca21), and environmental compounds (e.g., phenolic com- pounds, humic acids, and heavy metals). Other, more wide- spread inhibitors include constituents of bacterial cells, non- target DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including ap- proaches that have been used to overcome inhibition and fa- cilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been de- scribed, but these methods have received less attention. Problems sometimes occur with PCR, however (124). De- spite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reac- tion tube. Early evidence of exquisite sensitivity with mamma- lian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Con- sidering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most com- prehensive study of PCR inhibition, identifying inhibitory fac- tors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and in- organic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).
Oligonucleotide primers were designed for the PCR-based detection of the wec gene cluster involved in the biosynthetic pathway leading to the production of enterobacterial common antigen (ECA). Escherichia coli DNA was detected using wec A, wec E, and wec F gene primers. The wec A primers were specific for E. coli. The wec E and wec F primers enabled the detection of the most frequent species of the Enterobacteriaceae found in blood and urine specimens as well as in water. The sensitivity of the assay was approximately 1.2 x 102 bacteria/mL of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections. Their role in the routine testing of contamination in drinking water and food may prove to be very useful. The DNA of Enterobacteriaceae species is detected in a first step PCR, followed by specific identification of important pathogens like E. coli O157, Shigella spp., Salmonella spp., and Yersinia spp.
Identifying specific groups of mastitis pathogens by their growth on selective agars can help identify the pathogens that are present in mastitic milk samples. This article addresses issues that are essential in making good use of diagnostic procedures to improve udder health on dairies.
The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.
No nationwide studies of the incidence rate of clinical mastitis (IRCM) have been conducted in Canada. Because the IRCM and distribution of mastitis-causing bacteria may show substantial geographic variation, the primary objective of this study was to determine regional pathogen-specific IRCM on Canadian dairy farms. Additionally, the association of pathogen-specific IRCM with bulk milk somatic cell count (BMSCC) and barn type were determined. In total, 106 dairy farms in 10 provinces of Canada participated in the study for a period of 1 yr. Participating producers recorded 3,149 cases of clinical mastitis. The most frequently isolated mastitis pathogens were Staphylococcus aureus, Escherichia coli, Streptococcus uberis, and coagulase-negative staphylococci. Overall mean and median IRCM were 23.0 and 16.7 cases per 100 cow-years in the selected herds, respectively, with a range from 0.7 to 97.4 per herd. No association between BMSCC and overall IRCM was found, but E. coli and culture-negative IRCM were highest and Staph. aureus IRCM was lowest in low and medium BMSCC herds. Staphylococcus aureus, Strep. uberis, and Streptococcus dysgalactiae IRCM were lowest in the Western provinces. Staphylococcus aureus and Strep. dysgalactiae IRCM were highest in Québec. Cows in tie-stalls had higher incidences of Staph. aureus, Strep. uberis, coagulase-negative staphylococci, and other streptococcal IRCM compared with those in free-stalls, whereas cows in free stalls had higher Klebsiella spp. and E. coli IRCM than those in tie-stall barns. The focus of mastitis prevention and control programs should differ between regions and should be tailored to farms based on housing type and BMSCC.
JAGS is a program for Bayesian Graphical modelling which aims for compatibility with Classic BUGS. The program could eventually be developed as an R package. This article explains the motivations for this program, briefly describes the architecture and then discusses some ideas for a vectorized form of the BUGS language.
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