Veterinary Microbiology 146 (2010) 361–365

Short communication

Evaluation of Lawsonia intracellularis infection in a group of pigsin a subclinically affected herd from weaning to slaughter

Daniel Brandt a, Ute Kaim b, Wolfgang Baumgartner b, Michael Wendt a,*a Clinic for Swine and Small Ruminants, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germanyb Department for Pathology, University of Veterinary Medicine Hannover, Bunteweg 17, D-30559 Hannover, Germany

A R T I C L E I N F O

Article history:

Received 9 November 2009

Received in revised form 4 May 2010

Accepted 8 May 2010

Keywords:

Lawsonia intracellularis

Porcine proliferative enteropathy (PPE)

Subclinical infection

Pig

A B S T R A C T

The purpose of this study was to follow the course of a subclinical Lawsonia (L.)

intracellularis infection in a group of 60 pigs on a commercial farm from weaning to

slaughter. From 6 to 16 and at 26 weeks of age, rectal faecal samples and blood samples

were collected weekly from every pig for examination by PCR and blocking ELISA,

respectively. At corresponding times starting at 8 weeks of age, pigs were randomly

selected for necropsy (n = 51). Intestinal tissues were examined histopathologically and by

immunohistochemistry (IHC) for L. intracellularis antigen.

Infection with L. intracellularis showed a mainly subclinical course. Shedding of L.

intracellularis was detected by PCR in three pigs as early as 6 weeks of age and persisted up

until 14 weeks of age. In most pigs shedding of L. intracellularis was seen only for 1–2

weeks followed by a rapid serum antibody response. More than 50% of pigs had

seroconverted by week 10. At slaughter, 30.8% of investigated animals were still found to

be seropositive by ELISA.

Of the 60 study animals 39 were found positive by faeces PCR (65.0%), 49 animals were

found positive by serology (81.7%), and 35 pigs (68.6%) had positive results by IHC at

necropsy. All but one pig were found to be L. intracellularis infected by at least one of the

three methods (98.3%).

In conclusion, this is the first field study revealing the presence of prominent

histological lesions characteristic for L. intracellularis infection and associated positive

pathogen specific PCR and immunohistological results even in subclinically infected pigs.

Although intestinal alterations disappeared after 3–4 weeks, L. intracellularis was detected

by IHC for a longer time, especially in intestinal lymph nodes.

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1. Introduction

Infections by Lawsonia (L.) intracellularis, the causativeagent of porcine proliferative enteropathy (PPE), arewidely distributed in pig populations worldwide. Herdseroprevalence has been estimated at between 57% and100% (McOrist et al., 2003). In Germany 81.3% of 694

* Corresponding author. Tel.: +49 0511 856 7260;

fax: +49 0511 856 7684.

E-mail address: michael.wendt@tiho-hannover.de (M. Wendt).

0378-1135/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2010.05.021

investigated herds were found positive for L. intracellularis

in a serological survey (Wendt et al., 2006).L. intracellularis, can occur with typical signs of disease,

however, courses without clinical symptoms tend topredominate (Stege et al., 2004). Subclinical PPE producedin a challenge model with increasing doses of inoculumshowed no differences in the incidence of gross lesions, buta higher percentage of pigs with typical histologicallesions. All doses caused significant decreases in perfor-mance (Paradis et al., 2005).

The objective of this study was to monitor naturallyacquired L. intracellularis infection from weaning to the endof the fattening period in a herd with a suspected

D. Brandt et al. / Veterinary Microbiology 146 (2010) 361–365362

subclinical course of the disease (pigs without anorexia,diarrhoea or obvious wasting). This was accomplished byclinical investigation and serial testing of pigs for thepresence of L. intracellularis in faeces and for theproduction of specific serum antibodies and by examiningthe extent of histopathological lesions at different times ofinfection by post mortem investigation.

2. Materials and methods

Sixty pigs in a farrow-to-finish herd (120 sows) with aknown history of subclinical L. intracellularis infection wereselected after weaning at the age of 4 weeks and monitoredindividually from weaning to slaughter. In the nursery(360 places, 120 piglets weaned at a time), these pigs werehoused in one out of three rooms (6 out of 12 pens ingroups of 10 animals). At 10 weeks of age they were movedto the fattening unit (550 places), where the pigs were keptas a group in one room on slatted flooring.

Monitoring included clinical examination (faecal con-sistency: 0 = normal, 1 = pasty, 2 = semiliquid, 3 = watery)as well as blood and faeces sampling: at weekly intervalsfrom 6 to 16 weeks of age and once before slaughter (week26). Growth rate of the group at the age of 10 weeks wasinfluenced by an acute Actinobacillus pleuropneumoniae

(App) infection in the herd and was not further evaluated.Animals were treated with amoxicillin for 10 d (15 mg/kg BW/2 d), no other medication was given during thestudy.

Five pigs at a time were selected (simple randomsampling) and euthanised (weeks 8–16 as well as week 26,n = 50) for post mortem investigation. In addition, twostudy pigs were euthanised for animal welfare reasons, oneat week 10 (tibia fracture, no necropsy) and one at week 12(prolapse of rectum, necropsy).

2.1. PCR and serology

Faecal samples taken from the rectum were analysed bya PCR assay described by Jones et al. (1993) for detection ofL. intracellularis.

Blood samples were examined for the presenceof antibodies against L. intracellularis using a blockingELISA according to the manufacturer’s instructions(Enterisol1Ileitis-ELISA, bioScreen, Munster, Germany,officially registered in Germany by the Friedrich-Loeffler-Institute (FLI) Federal Research Institute forAnimal Health (Reg. No. FLI-B 390), not validated byreviewed publication).

2.2. Necropsy, histopathology and immunohistochemistry

(IHC)

The pigs underwent gross examination at necropsy, andsections from the small and large intestine were stained forhistopathology (HE) and, as well as Lnn. jejunales and Lnn.

ileocolici, were processed for immunohistochemical stain-ing (immunoperoxidase technique as described by Hsuet al., 1981; Guedes and Gebhart, 2003).

Histopathological lesions of nine gut sections werescored for different parameters (score 0–3 for epithelial

hyperplasia, villous atrophy, reduction of goblet cells,crypt abscessation, inflammatory cells, congestion ofblood vessels, haemorrhages, fibrinous membranes,necrosis; max. score for one section = 27) and scores forthe nine gut sections were added (max. score for onepig = 243).

Intestines were also investigated for other pathogens(Brachyspira hyodysenteriae, Brachyspira pilosicoli, Salmo-

nella, Escherichia coli) by usual culture techniques.

2.3. Statistical analysis

Correlation between the severity of histopathologicallesions and the time interval between first detection of L.

intracellularis infection and necropsy was tested usingSpearman’s rank correlation coefficient. The significance ofdifferences in average daily weight gain (week 8: group 1:still not infected (n = 46), group 2: L. intracellularis infected(n = 14)) and in the severity of histopathological lesions(detection of L. intracellularis infection less than 3 weeksbefore necropsy (group A, n = 22) and later than 3 weeks(group B, n = 23)) was tested using the Wilcoxon signed-rank test.

3. Results

3.1. PCR

In total, shedding of Lawsonia could be detected by PCRin 39 out of 60 animals (65%), one-third of the pigs had firstpositive results in weeks 9 and 10, highest shedding rateswere found between 22.5% and 27.3%. Detectable sheddingstopped after week 14 (Fig. 1). In most of the L.

intracellularis positive animals, shedding in faeces couldbe detected for 1 or 2 weeks only (n = 31; 17 pigs, whichwere necropsied after shedding had ended; 14 pigs, whereLawsonia could be detected up to post mortem investiga-tion and shedding could have lasted longer). There wereseven pigs where L. intracellularis could be found in faecesfor 3–5 weeks and only one pig shedding L. intracellularis

regularly for a longer period of 7 weeks.

3.2. Serology

Seroconversion against Lawsonia could be observed in49 out of the 60 pigs (81.7%), mostly between weeks 8 and11. Only three animals seroconverted after week 15. Thenumber of seropositive animals in the study groupincreased up to 72.7% in week 11, continuously decreasingafterwards (reaching 30.8% in week 26). Of the 49 pigs thatdeveloped antibody titers, L. intracellularis could not bedetected in the faeces of 15 of them at any time during thestudy (Fig. 1 and Table 1).

Sixteen (41.0%) of the 39 PCR-positive pigs showedseroconversion at the same sampling time when firstshedding of L. intracellularis was observed and 37.7%seroconverted 1–3 weeks after first detection of L.

intracellularis in faeces. Three animals showed antibodytiters before the first positive PCR result was obtained andfive pigs remained seronegative, although shedding waspresent.

Fig. 1. (A) Time of first L. intracellularis shedding (PCR, n = 39) and seroconversion (ELISA, n = 49) in a group of 60 pigs; (B) percentage of L. intracellularis

shedding pigs (PCR) and seropositive pigs (ELISA) at different sampling times.

D. Brandt et al. / Veterinary Microbiology 146 (2010) 361–365 363

3.3. Clinical symptoms

Examination of faecal consistency revealed only one pigshowing watery diarrhoea during the observation periodwhile in seven animals semiliquid faeces could beobserved at one (n = 6) or two points in time (n = 1). Theother 52 animals had solid to occasionally pasty faeces

Table 1

Comparison of results of three different methods (PCR, ELISA, n = 60; IHC

n = 51) for detecting L. intracellularis infection.

PCR+ PCR� ELISA, IHC

Total (n)

ELISA+ 34 15 49 (81.7%)

IHC+/� 24/8 2/6 26/14

ELISA? 4 2 6 (5.0%)

IHC+/� 3/1 2/0 5/1

ELISA� 1 4 5(13.3%)

IHC+/� 1/0 3/1 4/1

PCR total (n) 39 (65.0%) 21 (35.0%) 60 (100%)

IHC+/� total 28/9 7/7 35/16

+ = positive, ? = doubtful, and � = negative.

demonstrating a subclinical course of L. intracellularis

infection. From 4 to 8 weeks, 13 infected pigs had adecreased average daily weight gain in comparison to 46non-infected pigs (PCR/ELISA negative, 344� 92 g vs.394� 85 g). However, this difference was not significant(Wilcoxon test, p> 0.05).

3.4. Necropsy, histopathology and immunohistochemistry

(IHC)

Combining the results of IHC, PCR and ELISA investiga-tions, L. intracellularis infection was found in 59 out of 60pigs (98.3%) in total (details see Table 1).

The frequency of IHC positive pigs was high betweenweeks 8 and 13 (90.3%) and decreased in animals olderthan 13 weeks; at slaughter (week 26) none of the fiveinvestigated pigs were positive for L. intracellularis by IHC.

Gross intestinal lesions were detected only in oneanimal (fibrinous to necrotising enteritis). Histologicalexamination revealed typical lesions for PPE in 28 out of 51pigs in the ileum, caecum and Colon ascendens simulta-neously (crypt cell proliferation, goblet cell depletion,

Fig. 2. Correlation between the severity of histopathological lesions of the

intestine (total score, n = 45 pigs) and the time interval between first

detection of L. intracellularis infection (by PCR and/or ELISA) and necropsy

(y =�4.3985x + 35.598; r =�0.5753, p< 0.001).

D. Brandt et al. / Veterinary Microbiology 146 (2010) 361–365364

mitotic figures, crypt elongation). Five of these pigsadditionally had fibrinous to ulcerative lesions.

The severity of histopathological lesions (total score)correlated negatively with the time interval between firstdetection of L. intracellularis infection (by PCR and/orELISA) and necropsy (n = 45, r =�0.5753, p< 0.001; Fig. 2).Moderate to severe lesions (total score >10) were onlyseen in group A (necropsy �3 weeks after detection ofinfection), but not in group B (necropsy >3 weeks afterdetection of infection) (mean total score for all intestinalsections 28.77� 24.51 vs. 3.39� 2.05, p< 0.05).

In group A only 3 out of 22 pigs were negative by IHCand most of the pigs (n = 13) showed L. intracellularis in theapical cytoplasm of intestinal crypt cells and mononuclearcells in the epithelium of the small and large intestine aswell as in mononuclear cells in lymph nodes (only cryptcells positive: n = 5, only lymph nodes positive: n = 1). Ingroup B the number of IHC negative pigs was much higher(n = 12 out of 23) and positive IHC results were obtainedmainly in lymph nodes alone (n = 7).

E. coli (F4, positive for enterotoxins LTI und STII) wasisolated from the one pig demonstrating watery diarrhoeaand shedding of L. intracellularis at the time of necropsy. Noother pathogens such as B. hyodysenteriae, B. pilosicoli orSalmonella were detected at any time in the group.

4. Discussion

L. intracellularis infection in the investigated groupshowed a mainly subclinical course. Semiliquid (7/60) orwatery faeces (1/60) were detected (by rectal examina-tion) only in few individual pigs within the group and onlyfor a short period. Mixed infection with L. intracellularis andpathogenic E. coli was detected in the pig with waterydiarrhoea. During the study, watery diarrhoea was notobserved in any other groups of pigs within the herd. Theaverage daily weight gain was noted in this study up to anage of 8 weeks and decreased in L. intracellularis infectedanimals by 50 g/d. Although this effect was not statisticallysignificant, decreased average daily weight gain is knownto be an important consequence of subclinical L. intra-

cellularis infection (Stege et al., 2004; Paradis et al., 2005).

L. intracellularis infection spread effectively within thestudy group of pigs; between 30% and 90% of pigs wereinfected by week 10 in each of the six nursery pens. Morethan 50% of pigs showed seroconversion before leaving thenursery. Therefore, the onset and peaking of L. intracellu-

laris shedding was observed earlier in this herd than hasbeen reported in other studies (Stege et al., 2004; Jensenet al., 2005).

Detection of L. intracellularis in faeces was mainly ofshort duration and was followed by a rapid serologicalresponse in most of the pigs. Ten faecal PCR-positive pigswere tested negative by ELISA; this may have been due to avery short period between exposure to L. intracellularis andnecropsy without sufficient time for the development of adetectable antibody response.

Lower rates of detection by PCR (65.0%) in comparisonto ELISA (81.7%) can be explained by subclinical L.

intracellularis infection, since smaller amounts of L.

intracellularis in faeces as well as intermittent sheddingresulting in false negative PCR results would be expected(Guedes, 2004). PCR methods with a higher sensitivity(nested PCR, quantitative PCR) might have given betterresults (Jones et al., 1993; Nathues et al., 2007).

The number of seropositive pigs in the whole groupdecreased continuously from week 11 (72.7%) up toslaughter (30.8%). Other authors have reported higherpercentages of seropositive pigs at slaughter (Stege et al.,2004; van der Heijden et al., 2004). Low seroprevalence atslaughter may be related to subclinical infection withweaker immune responses in comparison to clinicaldisease, where peak serum titers are higher and longerlasting (Guedes, 2004). This may also be related to an earlyL. intracellularis infection in the herd with insufficient re-exposure to L. intracellularis during the fattening period,which would have led to a boosted response (Collins andLove, 2007). It cannot be ruled out that amoxicillintreatment in week 10 (App infection) may have influencedthe course of disease, although amocixillin has not beenrecommended to treat L. intracellularis infection (McOristand Gebhart, 1995).

Histopathological results demonstrated good correla-tions (r =�0.5753, p< 0.001) between the duration of L.

intracellularis infection and the degree of lesions in theintestine characteristic for PPE. Most severe histopatho-logical changes were observed 1–3 weeks after firstdetection of L. intracellularis infection (Fig. 2). Animalswith longer lasting L. intracellularis infection showed if atall only very mild proliferative lesions. These observationsare in accordance with previous studies (Jensen et al.,2005).

Our own findings of goblet cell hyperplasia as well as ofapoptotic bodies in epithelial cells in pigs with longerlasting L. intracellularis infection can be interpreted asrecovering stages of L. intracellularis infection (McOristet al., 1996).

Localisation of L. intracellularis detected by IHC corre-lated well with time of infection: L. intracellularis detectionin both the intestinal epithelium and intestinal lymphnodes corresponded to L. intracellularis infection notolder than 3–4 weeks, while L. intracellularis detection inlocal lymph nodes alone corresponded to longer lasting

D. Brandt et al. / Veterinary Microbiology 146 (2010) 361–365 365

infection. Pigs at slaughter can be L. intracellularis negativeby all three detection methods following recovery fromearly subclinical L. intracellularis infection and checks atthe slaughterhouse are often too late for meaningful PPEdiagnosis (Holyoake et al., 1994).

In conclusion, this is the first study showing that incases of natural L. intracellularis infection without anyappropriate treatment or prophylaxis against L. intracellu-

laris it is reasonable to hypothesise that in spite of asubclinical course, all animals in a group will suffer frominfection and that histopathological lesions characteristicfor PPE have to be expected. Although intestinal alterationsdisappear after 3–4 weeks, L. intracellularis can be detectedby IHC for a longer time, especially in intestinal lymphnodes.

Acknowledgments

We wish to thank Dr. Connie Gebhart, College ofVeterinary Medicine, University of Minnesota, St. Paul, USAfor providing the polyclonal antibodies against Lawsonia

intracellularis, the Institute for Microbiology, University ofVeterinary Medicine, Hannover, Germany for the culturalbacteriological examinations and Boehringer IngelheimVetmedica GmbH, Ingelheim, Germany for their financialsupport.

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