The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces

Ståhl, Marie; Kokotovic, Branko; Hjulsager, Charlotte Kristiane; Breum, Solvej Østergaard; Angen, Øystein

doi:10.1016/j.vetmic.2011.03.013

Cited by 50 publications

(42 citation statements)

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“…A number of additional laboratory investigations of the pigs not previously reported were performed using techniques previously described: colon tissue samples were examined for B. pilosicoli by fluorescent in-situ hybridisation (FISH) [8], jejunum content was examined for Rotavirus by ELISA, Salmonella spp. and E. coli [7], colon content was examined for Brachyspira spp [9], and faecal samples were subjected to qPCR testing for E. coli F4 and F18 genes [10]. …”

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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“…Theoretical pools calculated to contain less than 3.3 log 10 bacteria/g feces (qPCR limit of detection) 13 were classified as test negatives. All laboratory and theoretical pools were classified as L. intracellularis positives or negatives (dichotomized qPCR test results).…”

Section: Theoretical Poolmentioning

confidence: 99%

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

et al. 2014

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Abstract. Procedures in which biological specimens are mixed and tested as 1 sample (pooling) have been applied for various biological specimens and laboratory examinations. The objective of the current study was to investigate agreement between laboratory testing of fecal pools and theoretical values obtained by averaging test results from individual fecal samples in relation to a quantitative polymerase chain reaction (qPCR) test for Lawsonia intracellularis. Ten diarrheic and 10 normal fecal samples were submitted from each of 43 Danish swine herds (n = 860 fecal samples). Pools (n = 43), each containing 20 individual fecal samples from the same herd, were prepared in the laboratory by pooling 10% fecal phosphate buffered saline solutions. All pools and individual fecal samples were subjected to qPCR testing for L. intracellularis. The theoretical number of L. intracellularis in the pools was calculated as the mean number of bacteria from the 20 individual fecal samples contributing to each pool. Agreement between the laboratory testing of pools and theoretical calculations based on individual sample results was evaluated. Pooling resulted in fewer L. intracellularis-positive herds (41.9%) compared with testing 20 fecal samples (53.5%). Agreement between the laboratory and the theoretical pools for dichotomized test results was 100% (95% confidence interval: 91.8-100%). For the quantitative test results, Lin concordance correlation coefficient was 0.997. The mean difference between the laboratory testing and the theoretical values was not different from zero (mean difference = 0.039 log 10 bacteria/g feces; P = 0.26).

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“…The ISH, IHC, qPCR, and serology procedures were performed as previously described (Boye et al 1998;Boesen et al 2005;Enoe et al 2006;Jensen et al 2006;Hjulsager et al 2009;Stahl et al 2011).…”

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confidence: 99%