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ABSTRACT: 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 histopathological 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: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 03/2013; · 1.21 Impact Factor
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ABSTRACT: 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 log10 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 log10 copies/g faeces was not significantly associated with average daily gain of the pigs (P?<?0.5). CONCLUSION: The results suggest a potential application of a PCR quantifying L. intracellularis in growing pigs. Faecal dry matter content must be taken into consideration in interpretation of such test results.
Acta Veterinaria Scandinavica 09/2012; 54(1):58. · 1.00 Impact Factor
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ABSTRACT: Absolute quantification of Lawsonia intracellularis by real-time polymerase chain reaction (PCR) is now possible on a routine basis. Poor repeatability of quantification can result in disease status misclassification of individual pigs when a single fecal sample is obtained. The objective of the current study was to investigate overall variation within a day for fecal numbers of L. intracellularis bacteria determined by real-time PCR in growing pigs. From each of 30 pigs with an infection of L. intracellularis, 5 fecal samples were collected within 1 day. A total of 150 fecal samples were obtained and subjected to quantitative PCR (qPCR) testing. Mean fecal dry matter content was 14.3% (standard deviation = 4.5%). Two pigs (6.7%) alternated between being L. intracellularis qPCR positive and negative. For 28 pigs, the excreting levels of L. intracellularis were within the dynamic range of the qPCR assay at all sampling points. For these 28 pigs, the mean excretion level of L. intracellularis was 6.1 log(10) bacteria/g feces (standard deviation = 1.2 log(10) bacteria/g feces). The maximum observed difference between 2 fecal samples from the same pig was 1.1 log(10) bacteria/g feces. The average standard deviation for individual pigs was 0.27 log(10) bacteria/g feces. The average coefficient of variation within pigs was 0.04, ranging from 0.006 to 0.08. The results imply that absolute quantification of L. intracellularis by qPCR has acceptable repeatability within 1 day. However, a population-specific proportion of pigs alternating between positive and negative test results must be expected.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 07/2012; 24(5):968-70. · 1.21 Impact Factor
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ABSTRACT: Microwave drying as a procedure for determination of faecal dry matter in weaned pigs was evaluated and clinical relevant cut-off values between faecal consistency scores were determined. Repeatability and reproducibility were evaluated. Overall coefficient of variation was 0.03. The 95% confidence limits for any future faecal subsample examined by any operator in any replica were ± 0.85% faecal dry matter. Robustness in relation to weight of wet faeces was evaluated. The weight categories were 0.5, 1.0, 1.5, 2.0 and 3.0 g. Samples of 0.5 g gave significantly different mean faecal dry matter content compared to weighing of 1.0-3.0 g. Agreement with freeze-drying was evaluated. Lin's concordance correlation coefficient was 0.94. On average the faecal dry matter values was 1.7% (SD=1.99%) higher in freeze dried compared to micro waved samples. Non-parametric ROC analyses were used to determine optimal faecal dry matter cut-off values for clinical faecal consistency scores. The 4 consistency scores were score 1=firm and shaped, score 2=soft and shaped, score 3=loose and score 4=watery. The cut-off values were score 1: faecal dry matter content >19.5%, score 2: faecal dry matter content ≤ 19.5% and >18.0%, score 3: faecal dry matter content ≤ 18.0% and >11.3%, score 4: faecal dry matter content ≤ 11.3%. In conclusion, the microwave procedure has an acceptable repeatability/reproducibility and good agreement with freeze drying can be expected. A minimum of 1.0 g of wet faeces must be used for analyses. Faecal dry matter cut-off values between 4 different clinical consistency scores were determined.
Preventive Veterinary Medicine 07/2011; 100(3-4):163-70. · 2.05 Impact Factor
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ABSTRACT: Inter-observer agreement for assessment of faecal consistency in pigs was evaluated using a scoring system with 3 categories. In a pilot study, 3 observers performed an examination of faecal samples post-collection. The samples were obtained from pigs (12-13 weeks old) in 4 herds with a history of diarrhoea associated with Lawsonia intracellularis, Brachyspira spp. and/or Porcine Circovirus Type 2. Observer 1 examined all the faecal samples from the 4 herds. Observer 2 only examined the faecal samples from herds 1 and 2. Observer 3 only examined the faecal samples from herds 3 and 4. We observed a substantial agreement in faecal consistency scores between Observers 1 and 3 (kappa=0.64, 95% CI: 0.51-0.78). In contrast, only a fair agreement was observed between Observers 1 and 2 (kappa=0.24, 95% CI: 0.14-0.34). The variations in inter-observer agreement detected in the current study suggest that misclassification error can be a problem in studies assessing faecal consistency. Solutions may include developing a standardized system for scoring the consistency of pig faeces, calibration when more than one observer is involved in clinical studies and using a more objective measure of faecal consistency.
Preventive Veterinary Medicine 03/2011; 98(4):284-7. · 2.05 Impact Factor
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ABSTRACT: 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.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 07/2010; 22(4):487-94. · 1.21 Impact Factor
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ABSTRACT: The Danish surveillance-and-control program for Salmonella in slaughter pigs was introduced in 1995. The key element of the program is a quick and correct identification of herds with high seroprevalence. After 5 years, the classification scheme was evaluated--and a revision was made. Data from two Salmonella screenings including a total of 1902 slaughter pig herds were used. For each herd, information was available on Salmonella status based on both microbiology and serology. Based on analyses of these data, suitable changes in the scheme were identified and their effect estimated by use of data from the Danish Salmonella Database including all herds in 2000. The classification scheme has been adjusted on the following points. (1) The sampling has been simplified into 60, 75, or 100 samples per herd per year depending on herd size. This means more-precise estimates for the seroprevalence among smaller herds. (2) Herds with an annual kill <or=200 finishers will not form part of the surveillance; this leaves 1.6% of the slaughter pigs outside the surveillance scheme. (3) The cut-off for individual meat-juice samples has been reduced from OD% 40 to OD% 20--doubling the number of positive samples. (4) The results of the previous 3 months' serological samples will be weighed 0.6:0.2:0.2 (the immediate month counting three times as much as the previous months), and the weighed average is called the "serological Salmonella index" for slaughter pig herds. A herd with an increasing seroprevalence will be assigned to a higher Salmonella level more-quickly under the new scheme. (5) A herd will be assigned monthly to one of three levels. The limit between Levels 1 and 2 has been set to >or=index 40, and the limit between Levels 2 and 3 to >or=index 70. If the Danish swine producers are interested, a Level 0 may be introduced (consisting of seronegative herds as an indication of a negligible Salmonella prevalence). The classification scheme was introduced in August 2001.
Preventive Veterinary Medicine 02/2002; 53(1-2):133-46. · 2.05 Impact Factor
