Within-day repeatability for absolute quantification of Lawsonia intracellularis bacteria in feces from growing pigs (original) (raw)
Related papers
Journal of Veterinary Diagnostic Investigation, 2013
Quantitative polymerase chain reaction (qPCR) tests for detection and quantification of Lawsonia intracellularis in feces from pigs have been developed. The objective of the current study was to evaluate the diagnostic performance of a fecal qPCR test for detection of nursery pigs with L. intracellularis-associated proliferative enteropathy (PE) under field conditions. Furthermore, the diagnostic performance for different subpopulations of pigs was investigated, including pigs infected or noninfected with Porcine circovirus-2, Brachyspira pilosicoli, and Escherichia coli. The diagnostic performance was evaluated in terms of diagnostic sensitivity and specificity. Data from pigs originating from 20 herds with antibiotic treatment requiring diarrhea outbreaks from a prior study were reused. Before treatment, pigs were randomly selected for histological and immunohistochemical examination of intestinal segments and fecal quantification of L. intracellularis by qPCR. A total of 313 pigs (157 without diarrhea, 156 with diarrhea) were included in the statistical analysis, and 37 pigs (11.8%) were classified as PE positives (defined as proliferative histological lesions in combination with L. intracellularis demonstration by immunohistochemistry). Lawsonia intracellularis was detected by qPCR in feces from 91 pigs (29.1%). A nonparametric receiver operating characteristic (ROC) analysis provided an area under the ROC curve (0.93) and an optimal cutoff value of 4.8 log10 L. intracellularis bacteria/g feces. This cutoff provided a diagnostic sensitivity of 0.84 and diagnostic specificity of 0.93. The diagnostic sensitivity and specificity were significantly different between herds (P < 0.0001). Numerically, diagnostic sensitivity and specificity were different between subpopulations of pigs, but no significant differences were demonstrated.
Journal of Veterinary Diagnostic Investigation, 2010
Traditionally, diagnosis of Lawsonia intracellularis-associated proliferative enteropathy (PE) has depended on necropsy and histology. Since the establishment of the etiologic role of L. intracellularis, a number of specific polymerase chain reaction (PCR) assays have been developed for the detection of DNA in feces. The present article is a systematic review of peer-reviewed publications on the application of L. intracellularis-specific fecal PCR as an antemortem diagnostic test for histologic lesions of PE in pigs. Based on this information, a range of diagnostic sensitivities (36-100%) and specificities (50-100%) of the published tests was calculated. Validity and confidence limits of the estimates varied considerably. The positive and negative predictive values of 6 different PCR assays were calculated for PE prevalence of 15%, 30%, 45%, 60%, 75%, and 90%, using a histologic case definition of PE and based on the reported test sensitivities and specificities. The simulated predictive values suggested that applying the fecal PCR assay as a diagnostic test is more likely to overestimate than underestimate the number of pigs having histologic lesions of PE under field conditions. J Vet Diagn Invest 22:487-494 (2010) 488
Journal of Veterinary Diagnostic Investigation, 2014
Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Acta Veterinaria Scandinavica, 2012
Background: The objective of this study was to investigate the association between average daily gain and the number of Lawsonia intracellularis bacteria in faeces of growing pigs with different levels of diarrhoea. Methods: A longitudinal field study (n = 150 pigs) was performed in a Danish herd from day 29 to 47 post weaning. Every third day all pigs were weighed, subjected to a clinical examination and faecal samples were obtained. Faecal samples were subjected to dry matter determination and absolute quantification by PCR for L. intracellularis and porcine circovirus type 2 (PCV2). Association between average daily gain, faecal dry matter content, numbers of L. intracellularis bacteria and PCV2 genome copies in faeces was investigated in a multilevel mixed-effects linear model. Results: Increasing numbers of L. intracellularis log 10 bacteria/g faeces were significantly associated with decreasing average daily gain (P < 0.001). The association was decreasing with increasing faecal dry matter content (P < 0.01). The number of PCV2 log 10 copies/g faeces was not significantly associated with average daily gain of the pigs (P > 0.5).
Polish Journal of Veterinary Sciences, 2000
The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10 3 CFU/ml and 6.5x10 1 CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.
Molecular and Cellular Probes, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
2013
Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) and significantly more reproducible (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.
Acta Veterinaria Scandinavica, 2011
Background: Lawsonia intracellularis is a common cause of chronic diarrhoea and poor performance in young growing pigs. Diagnosis of this obligate intracellular bacterium is based on the demonstration of the microbe or microbial DNA in tissue specimens or faecal samples, or the demonstration of L. intracellularis-specific antibodies in sera. The aim of the present study was to evaluate a blocking ELISA in the detection of serum antibodies to L. intracellularis, by comparison to the previously widely used immunofluorescent antibody test (IFAT). Methods: Sera were collected from 176 pigs aged 8-12 weeks originating from 24 herds with or without problems with diarrhoea and poor performance in young growing pigs. Sera were analyzed by the blocking ELISA and by IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test and the results of the blocking ELISA was modelled against the IFAT test with a "2 dependent tests, 2 populations, no gold standard" model. Results: At the finally selected cut-off value of percent inhibition (PI) 35, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%. The positive predictive value was 0.82 and the negative predictive value was 0.89, at the observed prevalence of 33.5%.
BMC Veterinary Research, 2012
The study was designed to investigate correlation between histological findings of Lawsonia intracellularis in porcine cases of diarrhoea and the quantitative detection of Lawsonia intracellularis in faeces. A total of 156 pigs (10 to 70 days post weaning) with diarrhoea were randomly selected from 20 herds: The pigs were subjected to necropsy, histopathology, immunohistochemistry and faecal quantification of Lawsonia intracellularis by real time PCR. Results: The median Lawsonia intracellularis excretion was significantly higher in pigs with gross lesions of proliferative enteropathy (median excretion: 5.92 log 10 bacteria/g faeces) compared to pigs without gross lesions of proliferative enteropathy (median excretion: <3.3 log 10 bacteria/g faeces) (P<0.001). Spearman's correlation coefficient between the measureable PE lesions and L. intracellularis excretion was 0.50 (P<0.001). A significantly increasing trend in Lawsonia intracellularis excretion level for increasing proliferative enteropathy histopathology and immunohistochemistry scores was demonstrated (P<0.001; P<0.001). Spearman's correlation coefficient between the histopathology scores and L. intracellularis excretion was 0.67 (P<0.001). Spearman's correlation coefficient between the IHC scores and L. intracellularis excretion was 0.77 (P<0.001). Conclusions: The histological and quantitative PCR detection of Lawsonia intracellularis were correlated in pigs with diarrhoea. Overall the results suggest that clinically important levels for Lawsonia intracellularis excretion in faeces may be established. Such clinical threshold levels may be used in practice to confirm a diagnosis of Lawsonia intracellularis associated diarrhoea.