Pooling of porcine fecal samples for quantification of <i>Lawsonia intracellularis</i> by real-time polymerase chain reaction

Pedersen, Ken Steen; Johansen, Markku; Jorsal, Sven Erik Lind; Nielsen, Jens Peter; Bækbo, Poul; Angen, Øystein

doi:10.1177/1040638714524572

Cited by 11 publications

(8 citation statements)

References 14 publications

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“…The qPCR test results from the selected FAECAL-pigs were used. Pooling was simulated as previously described by calculating the mean number of L. intracellularis , B. pilosicoli and E. coli F4 and F18 genes based on the individual test results of the faecal samples contributing to each pool [11]. The selection of pigs from the FAECAL-pig data and classification of the test results in relation to each diarrhoea outbreak were repeated 10,000 times for each diagnostic procedure.…”

Section: Methodsmentioning

confidence: 99%

Herd diagnosis of low pathogen diarrhoea in growing pigs – a pilot study

et al. 2014

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BackgroundThe major indication for antibiotic use in Danish pigs is treatment of intestinal diseases post weaning. Clinical decisions on antibiotic batch medication are often based on inspection of diarrhoeic pools on the pen floor. In some of these treated diarrhoea outbreaks, intestinal pathogens can only be demonstrated in a small number of pigs within the treated group (low pathogen diarrhoea). Termination of antibiotic batch medication in herds suffering from such diarrhoea could potentially reduce the consumption of antibiotics in the pig industry. The objective of the present pilot study was to suggest criteria for herd diagnosis of low pathogen diarrhoea in growing pigs.Data previously collected from 20 Danish herds were used to create a case series of clinical diarrhoea outbreaks normally subjected to antibiotic treatment. In the present study, these diarrhoea outbreaks were classified as low pathogen (<15% of the pigs having bacterial intestinal disease) (n =5 outbreaks) or high pathogen (≥15% of the pigs having bacterial intestinal disease) (n =15 outbreaks). Based on the case series, different diagnostic procedures were explored, and criteria for herd diagnosis of low pathogen diarrhoea were suggested. The effect of sampling variation was explored by simulation.ResultsThe diagnostic procedure with the highest combined herd-level sensitivity and specificity was qPCR testing of a pooled sample containing 20 randomly selected faecal samples. The criteria for a positive test result (high pathogen diarrhoea outbreak) were an average of 1.5 diarrhoeic faecal pools on the floor of each pen in the room under investigation and a pathogenic bacterial load ≥35,000 per gram in the faecal pool tested by qPCR. The bacterial load was the sum of Lawsonia intracellularis, Brachyspira pilosicoli and Escherichia coli F4 and F18 bacteria per gram faeces. The herd-diagnostic performance was (herd-level) diagnostic sensitivity =0.99, diagnostic specificity =0.80, positive predictive value =0.94 and negative predictive value =0.96.ConclusionsThe pilot study suggests criteria for herd diagnosis of low pathogen diarrhoea in growing pigs. The suggested criteria should now be evaluated, and the effect of terminating antibiotic batch medication in herds identified as suffering from low pathogen diarrhoea should be explored.Electronic supplementary materialThe online version of this article (doi:10.1186/2046-0481-67-24) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Herd diagnosis of low pathogen diarrhoea in growing pigs – a pilot study

et al. 2014

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“…Such an approach has been shown to provide accurate results, while reducing time and labour requirements. Additionally, but perhaps more so in the veterinary world than in any clinical mass drug administration (MDA) programme, 'pooling' as a strategy may allow for a rapid estimation of drug efficacy or infection prevalence present in the herd based on microscopy results and subsequent faecal egg counts (FECs) [10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

“…In settings with low-intensity of infections, the majority of samples screened are expected to be negative [26]. The sensitivity of a given method might increase or decrease when pooling is recruited; increasing, when multiple 'weak' infections are combined in a single pool, so collectively the target of interest is detectable by qPCR and decreasing, when a single infected sample is 'buried' among uninfected ones, and subsequently diluted, hence undetectable by qPCR [11].…”

Section: Introductionmentioning

confidence: 99%

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

et al. 2019

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Background: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low-and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a 'pooling approach' can yield a low frequency of 'missed' infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in 'pools-of-five'. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.

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“…A Monte Carlo simulation also demonstrated that the pool size of 10 or 20 is preferred, when the DNA quantity levels ranged from 0.01 to 0.1 pg, compared to a pool size of 50 [14]. The reason for the poor detection of low Mptb DNA quantity in larger pool sizes may be due to the ‘dilution’ effect [13, 31]. This implies that the already low quantity of Mptb DNA in the low shedding group may become undetectable when pooled at a larger pool size.…”

Section: Discussionmentioning

confidence: 99%

Determining an optimal pool size for testing beef herds for Johne’s disease in Australia

Ly¹,

Dhand²,

Sergeant³

et al. 2019

PLoS ONE

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Bovine Johne’s disease (JD) is a chronic debilitating disease affecting cattle breeds worldwide. Pooled faecal samples are routinely tested by culture to detect Mycobacterium avium subsp. paratuberculosis (Mptb) infection. More recently, a direct high throughput molecular test has been introduced in Australia for the detection of Mptb in faeces to circumvent the long culture times, however, the optimal pool size for beef cattle faeces is not known. This study aimed to determine the optimal pool size to achieve the highest test sensitivity and specificity for beef cattle. Individual archived faecal samples with low, medium and high quantities of Mptb (n = 30) were pooled with faecal samples from confirmed JD negative animals to create pool sizes of 5, 10, 15 and 20, to assess the diagnostic sensitivity relative to individual faecal qPCR. Samples from JD-free cattle (n = 10) were similarly evaluated for diagnostic specificity. Overall, 160 pools were created, with Mptb DNA extracted using magnetic bead isolation method prior to Mptb-specific IS900 quantitative PCR (qPCR). The pool size of 10 yielded the highest sensitivity 73% (95% CI: 54–88%), regardless of the quantity of Mptb DNA present in the faeces. There was no significant differences between the four different pool sizes for positive pool detection, however, there was statistical significance between low, medium and high quantities of Mptb. Diagnostic specificity was determined to be 100%. The increase in pool size greater than 10 increased the chances of PCR inhibition, which was successfully relieved with the process of DNA dilution. The results of this study demonstrate that the pool size of 10 performed optimally in the direct faecal qPCR. The results from this study can be applied in future simulation modelling studies to provide suggestions on the cost-effective testing for JD in beef cattle.

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Pooling of porcine fecal samples for quantification of Lawsonia intracellularis by real-time polymerase chain reaction

Cited by 11 publications

References 14 publications

Herd diagnosis of low pathogen diarrhoea in growing pigs – a pilot study

Herd diagnosis of low pathogen diarrhoea in growing pigs – a pilot study

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

Determining an optimal pool size for testing beef herds for Johne’s disease in Australia

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