Routine q-PCR quantification of a number of virus infections is now well established in medicine and has recently been reviewed [12–14]. Application of qPCR for quantification of bacterial infections is less well established in routine diagnostics, especially in veterinary medicine. However, correlation between disease severity and qPCR quantification of bacterial infections in clinical samples from animals and humans has been reported for Borrelia burgdorferi, Mycoplasma genitalium, Brucella spp. , Helicobacter pylori, Mycobacterium leprae[19, 20], Mycoplasma gallisepticum, Streptococcus pneumoniae, Brucella melitensis, Haemophilus influenzae and Legionella penumophila. Further, clinical cut-off levels for bacterial load has been established for Streptococcus pneumoniae[22, 26], Haemophilus influenzae, Mycobacterium tuberculosis, Gardnella vaginalis and Atopobium vaginae.
In the current study a positive correlation between disease severity in terms of pathological findings and quantitative detection of L. intracellularis in faeces was demonstrated in pigs with diarrhoea. These results are in accordance with a previous report of correlation between L. intracellularis bacteria load in mucosal scrapings and the severity of intestinal lesions . In qPCR positive pigs the median L. intracellularis excretion level was higher in pigs with PE gross lesions compared to pigs without gross lesions. This suggests that quantification by qPCR might be applied for examination of presence or absence of gross lesions of PE. The extent of PE gross lesions and L. intracellularis excretion levels were apparently not correlated. However, this association should be further investigated because of the low number of pigs with gross lesions in the current study.
In contrast, increasing histopathology/IHC scores were correlated to increasing L. intracellularis excretion levels. One interesting aspect of the observed observations is that demonstration of L. intracellularis in faeces is not evident of ileitis but in case of ileitis high excretion levels of L. intracellularis can be expected. The applied study design did not take progression of L. intracellularis infection into consideration. The obtained results are only relevant for the association between gross pathology, histopathology, IHC and quantification of L. intracellularis by qPCR at the time of faecal sampling. Correlation over time and progression of L. intracellularis excretion during an infection should be performed in a longitudinal study design. Factors that potential could have influenced the results are the intermitted shedding of L. intracellularis previously described  and non-homogeneous distribution of L. intracellularis in faeces and/or intestinal tissue. Quantification of L. intracellularis in faeces has been reported to have an acceptable within day repeatability  suggesting that non-homogeneous distribution of L. intracellularis in faeces is not a major bias in the current study. Some of the qPCR negative animals were probably false negatives because of intermitted shedding caused by a L. intracellularis excretion level close to the qPCR’s limit of detection as previously described . This aspect has potentially introduced misclassification bias in the study. However, it is most likely only low excreting pigs that were classified as false negatives introducing only minor bias in the ranking of animals, the statistical analysis and the results. Inclusion of multiple standardized intestinal segments and inclusion of any gross lesions in the histological examinations have probably decreased any effect of a potential non-homogeneous distribution of L. intracellularis in the intestinal tissue.
Other factors including intestinal infections could potentially influence the reported associations. The reported lesions (IHC, proliferative lesions) are very specific for L. intracellularis. Therefore it seems very unlikely that other factors would have a confounding effect between these lesions and the faecal L. intracellularis excretion. However it is possible that interactions exists providing different absolute quantitative associations between L. intracellularis and the lesions depending on simultaneous infections or other factors. The number of observations in the current dataset did not allow for investigation of this aspect, which should be explored before clinical relevant threshold levels can be used in practice.