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The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge
Veterinary Microbiology 103 (2004) 35–45 www.elsevier.com/locate/vetmic
The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge Henriette T. Boesena,*, Tim K. Jensena, Anja S. Schmidta, Bent B. Jensenb, Søren M. Jensena, Kristian Møllera a
Danish Institute for Food and Veterinary Research, Bu¨lowsvej 27, DK-1790 Copenhagen V, Denmark b Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark Received 26 November 2003; received in revised form 17 May 2004; accepted 14 June 2004
Abstract The objective of this investigation was to study if different feeding strategies influence experimental infections of pigs with Lawsonia intracellularis, the causative agent of proliferative enteropathy. In three sequential trials, a total of 144 weaned pigs were fed five different diets all made from a standard diet based on wheat and barley as carbohydrate source and soybean as protein source. The five diets were: a standard diet (fine ground and pelleted), the standard diet fed as fermented liquid feed, the standard diet added 1.8% formic acid, the standard diet added 2.4% lactic acid and a diet similar to the standard diet (made from the same ingredients), but fed coarse ground. Twenty-four pigs on each diet were orally inoculated with L. intracellularis and growth performance and faecal excretion of bacteria were monitored. Twenty-four pigs fed the standard diet were included as not experimentally infected controls. Pigs in the first two trials were sacrificed 4 weeks post-inoculation, whereas animals in the third trial were sacrificed after 5 weeks. Pigs in all experimentally infected groups excreted L. intracellularis. The fermented liquid diet delayed the excretion of L. intracellularis and furthermore, pigs fed the standard diet supplemented with lactic acid had limited pathological lesions when the intestines were examined 4 weeks after inoculation. The growth performance was reduced in pigs experimentally challenged with L. intracellularis, however the prevalence and severity of diarrhea was limited. # 2004 Elsevier B.V. All rights reserved. Keywords: Lawsonia intracellularis; Proliferative enteropathy; Feed; Experimental infection; Pigs
1. Introduction Lawsonia intracellularis is an obligate intracellular bacterium causing proliferative enteropathy (PE). The * Corresponding author. Tel.: +45 72 34 60 00; fax: +45 72 34 60 01. E-mail address: [email protected] (H.T. Boesen).
infection causes diarrhea, retarded growth and/or sudden death in pigs and is one of the most economically important diseases in the swine industry worldwide (Lawson and Gebhart, 2000). In a recent Danish study L. intracellularis was detected by PCR in 93.7% of 79 randomly selected herds (Stege et al., 2000). The disease is characterized by adenomatous proliferation of epithelial cells infected by the bacterium and is
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.06.008
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grossly seen as a thickening of the intestinal mucosa in the aboral part of the small intestines, particularly the ileum, but may involve cecum and colon as well (Lawson and Gebhart, 2000). Antimicrobial growth promoters (AGP) has been used in many countries to control subclinical infections with L. intracellularis, however in Denmark the use of AGP was terminated in 1999 and since then the use of therapeutic antibiotic for pigs has increased, especially antibiotics effective of controlling L. intracellularis infections (WHO Report, 2003). Alternative methods to control the intestinal pathogen are therefore relevant to investigate. Diet fed to healthy animals is an important factor, which can regulate the microbial flora of the intestines (Leser et al., 2000). Different diets have been shown to affect the intestinal function in pigs by several mechanisms. Recently it has been shown, that feeding pigs a diet consisting of cooked rice and animal protein reduces the severity of the experimental infection with Brachyspira pilosicoli (Lindecrona et al., 2003b) and B. hyodysenteriae (Siba et al., 1996). Fermented liquid feed decreases the bacterial population throughout the gastrointestinal tract of pigs (Mikkelsen and Jensen, 1998; Højberg et al., 2003), and it is demonstrated to have a preventing effect on the development of swine dysentery (Lindecrona et al., 2003a). Coarse ground non-pelleted feed affects intestinal morphology in terms of an increase in height and volume of crypts (Brunsgaard, 1998) and has in a previous study been shown to reduce the prevalence of L. intracellularis (Stege et al., 2001). Both fermented liquid feed, organic acid and coarse non-pelleted feed increase the concentration of organic acids and lower the pH in the stomach. Consequently, enteric bacteria like Salmonella spp. and Escherichia coli are killed or reduced in number before reaching the parts of the gastrointestinal tract in which they will normally proliferate (Jensen et al., 2003a). Lactic acid and formic acid have different effects on the gastrointestinal microbiota. Lactic acid tends to increase the number of lactic acid bacteria, increase the population of yeast and to decrease the number of coliform bacteria along the gastrointestinal tract (Maribo et al., 2000). Formic acid, on the other hand, decreases the number of lactic acid bacteria, coliform bacteria and yeast along the gastrointestinal tract (Maribo et al., 2000; Canibe and Jensen, 2001).
In the current study, the effects of fermenting, acidifying and grinding the feed on L. intracellularis colonization and the development of PE after experimental challenge with L. intracellularis were investigated. Important evaluation parameters were growth performance and faecal excretion of bacteria, whereas induction of diarrhea was regarded a less critical parameter as many L. intracellularis infected herds in Denmark only have a history of unthriftiness among the pigs.
2. Material and methods 2.1. Animals and treatment groups A total of 144 (three trials of 48) pigs (Danish Landrace/Yorkshire/Duroc) of mixed sex, 6–7 weeks of age and weighing 10.4 2.1 kg (mean S.D.) (range 6.4–15.5 kg) were purchased from two high health herds known to be free of B. hyodysenteriae and L. intracellularis after medicated eradication program (Johansen et al., 2002). The absence of L. intracellularis were confirmed by PCR analysis (Lindecrona et al., 2002) of 50 faeces samples from pigs at different age group collected from the herds twice (with 2 weeks interval) prior to the experiments. One week before the end of the experiments faecal samples were tested for the presence of Salmonella spp. and pathogenic Brachyspira spp. by bacteriological culture (Stege et al., 2000). Seven pigs (four pigs in trial 2 and three pigs in trial 3) died or were euthanized due to illness not related to L. intracellularis during the study. 2.2. Experimental design The pigs were allocated to six separate pens and each pen contained eight pigs in each sequential trial. Five groups of pigs were offered one of five diets and each group was infected with L. intracellularis after 1 week on the diet. The sixth pen housed groups of eight control animals not exposed to L. intracellularis (not experimentally infected, N-INF). The pens were separated from each other by ‘‘open-mesh wire’’ walls, which permitted some contact between the animals. The pens had slatted floors and no bedding was provided. The N-INF group was isolated from the infected groups
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by a gangway and attendants always first sampled from the N-INF pigs before sampling from the infected groups. The whole stable with pens was formalin sterilized prior to introduction the pigs and no other pigs were kept within the premises during the experimentation. All procedures of animal handling and experimentation were performed under veterinary supervision and according to recommendations by the Danish Committee of Animal Experimentation.
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by grinding through a 5 mm sieve in the same mill. The fermented liquid feed was prepared in an 80-L closed tank with continuous stirring as described by Mikkelsen and Jensen (1998). In short, the standard diet was mixed with water at a ratio of 1:2.5 (w/w). The feed was allowed to pre-ferment at 24 8C for 3–4 days before onset of the experiment. At each feeding, one-quarter of the content was taken from the tank, and the tank was refilled with an equivalent amount of new food and water. The pigs were fed the experimental diets ad libitum.
2.3. Diets 2.4. Experimental challenge Five different diets all made from a standard diet based on wheat and barley as carbohydrate source and soybean as protein source (Table 1) were used in the present experiment. The five diets were: STD, standard diet (fine ground and pelleted); FLF, the standard diet fed as fermented liquid feed; FAC, the standard diet added 1.8% formic acid; LAC, the standard diet added 2.4% lactic acid and COARSE, a diet similar to the standard diet (made from the same ingredients, Table 1) but fed coarse ground. The NINF groups were fed STD. The fine pelleted diets (STD, FLF, FAC and LAC) were ground through a 2 mm sieve while the coarse ground diet was obtained
Table 1 Composition of the diet Item
Feed (g/kg)
Wheat Barley Dehulled toasted soybean meal Rapeseed meal Sunflower meal Animal fat Calcium carbonate Dicalcium phosphate Sodium chloride L-Lysine, 40% Vitamin and mineral premixa DL-Methionine, 40% Threonine, 50%
360.8 360.7 122.9 80.0 20.0 25.3 8.0 11.5 3.9 2.9 2.0 1.2 0.8
a
Supplied per kilogram of diet: 2200 IU Vitamin A; 500 IU Vitamin D3; 30,000 mg Vitamin E; 1100 mg Vitamin K3; 1100 mg Vitamin B1; 2000 mg of Vitamin B2; 1650 mg of Vitamin B6; 5500 mg D-pantothenic acid; 11000 mg niacin; 27.5 mg biotin; 11 mg Vitamin B12; 25,000 mg FeSO4x7H2O; 40,000 mg ZnO; 13,860 mg MnO; 10,000 mg CuSO4x5H2O; 99 mg KI; 150 mg Na2SeO3.
PE-affected small intestines collected from two farms were utilized for the preparation of the L. intracellular homogenate inoculum. Frozen (80 8C) intestines were thawed and the mucosa scraped using a sterile scalpel. The scraped mucosa was combined with sucrose–potassium–glutamate (SPG, pH 7.0) and homogenized in a blender for 2 min. The inoculum was kept at 4 8C until use on the same day. L. intracellularis was quantified in the homogenate by making serial 1:10 dilutions of the homogenate in SPG with 5% fecal calf serum. Six-well glass slides were coated with 10 ml of each dilution, dried at room temperature, fixed in methanol, and stained by an indirect immunofluorescence method (Jensen et al., 1997) using a monoclonal antibody specific for L. intracellularis (Law1-DK) (Boesen et al., 2004). The number of fluorescent bacteria was counted in 10 view fields at 40 objective representing 1/25 of the total well. Pigs were fasted for 24 h before inoculation. They were infected via stomach tube by administering approximately 25 ml of L. intracellularis homogenate containing 3.6 1.2 108 bacteria/ml (mean S.D.). The N-INF groups received 25 ml SPG. 2.5. Monitoring of the infection Faecal samples were collected from each pig 24 h prior to the L. intracellularis inoculation and then every 2–3 days during the study. Samples were analyzed by real time PCR for detection of L. intracellularis DNA (Lindecrona et al., 2002). When L. intracellularis was detected from the same pig in two consecutive faecal samples, the pig was con-
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sidered to be excreting L. intracellularis in the period between these two samples. The pigs were weighed every 7 days throughout the experimental period to determine the growth rate. The pigs were observed daily for clinical signs of PE, in particular diarrhea. At the end of the experimentation (4 or 5 weeks (trial 3) after challenge) the animals were sacrificed by means of captive bolt pistol followed by pitching and exsanguination. The animals were necropsied and tissue samples were taken from ileum, caecum and top of the spiral colon for histopathologically examination. The samples were placed in 10% neutralbuffered formalin. After fixation the material was dehydrated, embedded in paraffin, sectioned at 3 mm and immune stained with a L. intracellularis specific monoclonal antibody (Law1-DK) according to Jensen et al. (2003b). Briefly, after deparaffinization in xylene the sections were rehydrated and treated with 0.6% H2O2 in Trisma base buffer (TBS) (50 mM Tris, 150 mM NaCl, pH 7.6) for 20 min. Then washed 3 min 5 min in TBS followed by incubation in a moisture chamber for 30 min with the MAb in TBS. Washed 3 min 5 min in TBS before incubation with EnVision+TM (DAKO, Glostrup, Denmark) for 30 min. After 3 min 5 min wash in TBS the reaction was developed for 15–20 min with a solution of 3-amino-9-ethylcarbozole (KemEnTec, Copenhagen, Denmark), counterstained with Mayer’s haematoxylin and mounted with Glycergel (DAKO). The immune stained sections were examined for the presence of L. intracellularis antigen in the crypt
and surface epithelium of the intestines as well as within subepithelial macrophages. The animals were scored according to the severity and extension of the lesions: (+) focal detection of L. intracellularis antigen within macrophages only, (++) focal detection of L. intracellularis within crypt epithelium (focal PE), (+++) multifocal detection of L. intracellularis infected crypts (PE), (++++) detection of L. intracellularis infected crypts diffusely within the mucosa (PE). Only pigs surviving during the whole experimental period were included in the weight, faecal excretion and immunohistochemistry data set. Table 2 includes the number of pigs included in the data material.
2.6. Data analysis Data were statistically analyzed by one-way analysis of variance (ANOVA) followed either by Dunnett’s multiple comparison test for comparing mean duration of faecal excretion between pigs feed STD and pigs feed the modified diets or by Tukey’s multiple range test for comparison of average daily weight gain. Results are expressed as mean standard error of the mean. Data analyses were performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA, USA. Power analyses and effect size index were carried out using G*power, http://www.psycho.uni-duesseldorf. de/aap/projects/gpower/index.html.
Table 2 Faecal excretion of L. intracellularis after experimental challenge and actual number of inoculated pigs given different diets in triplicate trials Dieta
Trial 1/2/3 Number of pigs inoculated or not experimental infectedb
Excretion first detected after day 5 post-exposure (days after inoculation)
Pigs that excrete L. intracellularis (no. of pigs)
STD FLF FAC LAC COARSE
8/8/8 8/8/7 8/8/8 8/7/8 8/5/7
8/8/8 10/10/10 8/6/8 6/8/10 8/6/8
7/8/4 8/7/7 8/7/8 7/2/7 8/3/7
N-INF
8/8/7
15/27/0
5/3/0
a
STD: fine ground and pelleted standard diet; FLF: fermented liquid feed; FAC: standard diet with formic acid; LAC: standard diet with lactic acid; COARSE: coarse ground standard diet; N-INF: not experimental infected pigs fed the STD diet. b Pigs inoculated with L. intracellularis or not experimental infected, which survived through the entire experiment period. Numbers represent sequential trial results, e.g. 8/7/8 represents 8, 7 and 8 animals in sequential trials.
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3. Results 3.1. Faecal excretion of L. intracellularis following experimental challenge The percentages of pigs that excrete L. intracellularis in the different diet groups are presented in Fig. 1. Two to three weeks after inoculation 65–90% of the pigs from all diet groups excreted L. intracellularis. Four weeks after inoculation 40–65% of the pigs still excreted L. intracellularis. Besides excretion of L. intracellularis in faeces immediately after inoculation, the excretion of bacteria was not detectable until 10 days after inoculation in the group fed the FLF (Fig. 1 and Table 2). In all other diet groups the excretion was initiated already 6–8 days after inoculation (except for diet group LAC in trial 3 which initiated excretion after 10 days). In Table 2 the number of pigs experimentally inoculated with L. intracellularis, which survived throughout the study are listed for each trial together with the number of pigs excreting L. intracellularis. Of the experimentally infected pigs, 14% did not excrete L. intracellularis at any time throughout the study. Eight pigs from the N-INF groups excreted L. intracellularis at the termination of the observation period (trials 1 and 2, Fig. 1 and Table 2). The mean duration of faecal excretion in pigs that
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excreted L. intracellularis (excluding data immediately post-inoculation) is illustrated in Fig. 2. In the different diet groups the mean duration of faecal excretion varied from 11.7 to 15.6 days. The mean duration of faecal excretion in the FLF fed group was significantly lower than in pigs fed the STD (P < 0.05). No significant difference in duration of bacteria excretion was observed between the STD group and any of the other diet groups. The power of the test was 67% with an effect size index of 0.31. Calculations were based on an accumulation of data from the three trials, as there was a good accordance in faecal excretion between the different trials. 3.2. Growth rate Pigs were weighed throughout the experimental period and the average weight for pigs on the different diets are presented in Fig. 3. For the first 2 weeks after inoculation the average weight gain was 215 86 g/ day (mean S.D.) for pigs in all the tested diet groups. Differences in growth rate between the L. intracellularis experimentally infected diet groups and the N-INF group initiated 2 weeks after inoculation, and the average daily weight gain from that time point to sacrifice is illustrated in Fig. 4. In an interval of 2–4 weeks after the time of inoculation the
Fig. 1. The faecal excretion after experimental challenge with L. intracellularis in pigs offered various diets. Values are total number of pigs excreting L. intracellularis in three trials in percentage of the total number of pigs inoculated. The pigs were fed either STD, n = 24 (~); FLF, n = 23 (&); FAC, n = 24 (~); LAC, n = 23 (); or COARSE, n = 20 (^). Not experimentally infected pigs were fed the STD diet, n = 23 (*).
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Fig. 2. The duration of faecal excretion after experimental challenge with L. intracellularis in pigs offered various diets. Values are mean excretion days + S.E.M. of pigs that at any time of the experiment excreted L. intracellularis. Groups with (*) differ from the STD group at P < 0.05 (by one-way analysis of variance with Dunnett’s adjustment).
average daily weight gain of the N-INF group was superior compared to the individual experimentally infected groups in the same time interval. This difference was, however, only significant (P < 0.01) when comparing the experimentally infected pigs feed the STD or FLF with the N-INF group. The five diet groups experimentally infected with L. intracellularis showed no difference in terms of weight gain. The power of the study was 90% with an effect size index of 0.36. Calculations were based on an accumulation
of data from the three trials, as there was a good accordance in weight gain between the different trials. 3.3. Clinical outcome The clinical signs of L. intracellularis infection in terms of diarrhea were very limited, even in pigs excreting bacteria for longer periods. Except from the pigs with E. coli diarrhea (see Section 3.4) a few pigs from each group had signs of loose faeces,
Fig. 3. The average weight of pigs offered various diets and experimentally challenged with L. intracellularis. The pigs were fed either STD, n = 24 (~); FLF, n = 23 (&); FAC, n = 24 (~); LAC, n = 23 (); or COARSE, n = 20 (^). One group of pigs was fed the STD and left not experimental infected; N-INF, n = 23 (&). Values are mean weight S.E.M.
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Fig. 4. The average daily weight gain from 2 to 4 weeks after experimental challenge with L. intracellularis in pigs offered various diets. The results are mean weight gain + S.E.M. Groups sharing same letters are significant different from each other at P < 0.01 (by one-way analysis of variance with Tukey’s adjustment).
but we seldom observed diarrhea (watery and grey faces). 3.4. Other infections Three weeks after inoculation with L. intracellularis faecal samples were tested negative for the presence of B. pilosicoli, B. hyodysenteriae and Salmonella spp. by bacteriological culturing. In the second and third trial, some pigs suffered from E. coli associated diarrhea in the second week of experimentation. As especially pigs from diet groups fed LAC and COARSE in trial 2 were affected both groups were treated twice i.m. with 225 mg amoxicillin (Clamoxyl1). Seven pigs (four pigs in trial 2 and three pigs in trial 3) died or were euthanised due to the E. coli infection. 3.5. Necropsy and histopathology Grossly thickening of intestinal mucosa in the aboral part of the small intestine was observed 4 weeks after inoculation in 13–38% of the pigs fed the STD, FLF, FAC and COARSE, but not in pigs fed the LAC. In Fig. 5 an ileum with typically thickened mucosa is compared to ileum from a N-INF pig. In trial 3, where the pigs were kept alive for an extra week, a similar grossly thickening of the intestinal
mucosa was only seen in one and two pigs from diet groups LAC and COARSE, respectively (data not shown). Large intestinal gross lesions were not observed in any pigs. In trials 1 and 2, a few pigs in all diet groups showed ulceration of pars oesophagea of the stomach. Extragastro-intestinale lesions were not observed. Diffuse PE with L. intracellularis in the apical cytoplasm of immature crypt enterocytes was demonstrated in the most severely affected ilea together with superficial necrosis. In the ileum from some pigs without gross lesions, L. intracellularis antigen was detected within the apical cytoplasm of surface enterocytes as well as in subepithelial macrophages between normally appearing crypts (Fig. 6). The extent of L. intracellularis infection in the large intestines was only minor compared to the lesions in ileum (data not shown). Fig. 7 illustrates the scoring of positive immune staining for L. intracellularis in ileal sections of pigs inoculated with the bacterium and sacrificed 4 or 5 weeks post-exposure. The degree of infection at 4 weeks after inoculation (trials 1 and 2) tended to be less severe in the diet group fed LAC, as L. intracellular antigen was detected within subepithelial macrophages only. Diet groups STD, FLF, FAC and COARSE all included pigs with proliferative immature enterocytes infected by L. intracellularis in the crypts at the time of sacrifice (Fig. 7a and b). Five
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Fig. 5. Gross appearance of an ileum with diffuse PE from a pig challenged with L. intracellularis (COARSE) (top) compared to the ileum from a N-INF control (bottom). The mucosa of the affected ileum is evidently thickened and ridged.
Fig. 6. Ileum from a pig sacrificed 4 weeks after experimental challenged with L. intracellularis. Immunohistochemical detection of L. intracellularis antigen (brownish-red) diffusely within large, subepithelial macrophages and as small rods in the apical cytoplasm of surface enterocytes (inserted). The adjacent crypts are lined by mature enterocytes including goblet cells.
weeks after inoculation (trial 3), fewer pigs showed PE with infected immature enterocytes, but L. intracellularis antigen was demonstrated within subepithelial macrophages (Fig. 7c). In seven of the eight N-INF pigs from trials 1 and 2, which excreted L. intracellularis during the experiment, bacteria were detected in proliferative immature enterocytes. Furthermore, L. intracellularis were present in sections of ileum of four N-INF pigs from trial 3, although the pigs did not excrete the bacterium (results not shown).
4. Discussion This study examined the influence of different diets on the colonization of L. intracellularis in weaned pigs after an experimental challenge. Diet type and feeding systems have not been identified as risk factors in a questionnairy survey on British pig farms (Smith et al., 1998). However, this type of investigation may have limitations as to exact knowledge of the different management parameters, content of feed and exact diagnosis of pigs, and we therefore considered it
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Fig. 7. Scoring of positive immune staining for L. intracellularis in ileal sections after experimental challenge in pigs offered various diets. Values are total numbers of pigs found positive by immunohistochemistry in percentage of the total number of pigs inoculated. Pigs were either sacrificed 4 weeks ((a) trial 1; and (b) trial 2) or 5 weeks ((c) trial 3) after inoculation. Scoring: (+) focal detection of L. intracellularis antigen within macrophages only, (++) detection of L. intracellularis focally within enterocytes (focal PE), (+++) detection of L. intracelularis multifocally within enterocytes, PE, (++++) detection of L. intracellularis diffusely within enterocytes, PE.
relevant to study the influence of diet on L. intracellularis colonization in experimentally infected pigs. The mean duration of faecal excretion of L. intracellularis was significantly lower in pigs consuming a fermented liquid standard diet compared to pigs
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fed a non-fermented diet. In previous experimentally infection studies with two other intestinal pathogens, B. hyodysenteriae and B. pilosicoli, the effect of similar diet groups were tested (Lindecrona et al., 2003a,b). In these studies there were a preventing effect of the fermented diet on the B. hyodysenteriae infection, but no effect on experimental infection with B. pilosicoli. Feeding fermented liquid feed reduces pH to between 4 and 5 in the stomach and the density of coliform bacteria is decreased throughout the gastrointestinal tract in pigs (Mikkelsen and Jensen, 1998). The acidity of the feed might impair the survival of L. intracellularis and explain the reduced time of L. intracellularis excretion in this diet group. Additionally, fermented liquid feed contains high number of lactobacilli and high concentration of lactic acid, and feeding FLF to pigs leads to higher number of lactobacilli in the small intestine (Mikkelsen and Jensen, 1998), which may delay the colonization of L. intracellularis. The addition of organic acids to the feed, either as lactic acid or as formic acid, had no reducing effect on the mean duration of faecal excretion of L. intracellularis, however, as the study only had 67% power to find a statistically significant difference, we can not necessarily conclude that the treatment was ineffective. To reach a firm conclusion on the absence of effect the sample size will have to be increased. In the present investigation, the pigs fed the diet with lactic acid had fewer lesions in the intestine 4 weeks after inoculation compared to the pigs fed the STD. Pigs fed both diets supplemented with organic acids had a slightly improvement of growth rate compared to the pigs fed the STD. The use of lactic acid is known to increase the concentration of lactic acid and to decrease pH in stomach content of pigs and consequently to kill coliform bacteria like E. coli and Salmonella spp. (Jensen et al., 2003a). A similar effect of the LAC on the gastrointestinal ecosystem may also explain the reduction of the infection with L. intracellularis. It has previously been reported that home-mixed diet is associated with reduced prevalence of L. intracellularis (Stege et al., 2001). In the present study, feeding coarse ground, non-pelleted feed, which mimicked a home-mixed diet, did not reduce the infection with L. intracellularis, compared with the fine-ground and pelleted standard feed. Differences in observations may be a result of the setup of the investigations. In the present study, the infections were experimental whereas the study by Stege and
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coworkers was based on a questionnaire where natural infections of L. intracellularis in 79 randomly selected herds were investigated. In the second trial of the present investigation, two diet groups of pigs (LAC and COARSE) suffered from an outbreak of E. coli associated diarrhea shortly after the L. intracellularis inoculation, which compelled us to treat pigs in these groups with amoxicillin. This antibiotic may have had an effect on L. intracellularis either directly as L. intracellularis is inhibited by the related ampicillin (McOrist et al., 1995) or indirectly by an effect on the general bacterial community in the intestine, which may affect the degree of the infection. L. intracellularis has previously been shown to be dependent on the presence of other enterobacteria, as an infection could not be experimentally established in gnotobiotic pigs (McOrist et al., 1993). These facts may explain the low infection rate in the amoxicillintreated groups, however exclusion of data obtained in these two diet groups does not change the statistical assessment of the presented influence of diet on L. intracellularis infections. The in vitro maintenance of L. intracellularis is difficult, and we therefore conducted our infection model using infected intestinal mucosa from PE-affected pigs. PE has successfully been experimentally reproduced in pigs inoculated with infected intestinal mucosa in several studies (Lomax et al., 1982; Mapother et al., 1987; Guedes and Gebhart, 2003), and our experimental model resulted in mild to moderate clinical symptoms. Pigs excreted L. intracellularis starting 1 week postinoculation, but the infection was not associated with mortality as in the infection model established by Guedes and coworkers (Guedes et al., 2002). The differences in morbidity of experimental infection models may be the result of different pathogenecity of the bacteria used for challenge. However, it is noticeable that all infected groups in the present study had a lower average weight gain than the N-INF group. All together, the present infection model yielded a morbidity and severity, which is in accordance with the current Danish field situation of the disease. Furthermore, lesions found in several of the pigs sacrificed 4 weeks post-challenge showed evident PE, similar to that described in natural as well as in experimental infection (Jensen et al., 1997; McOrist et al., 1993). When the intestines were histopathologically examined some pigs revealed only L. intracellularis
antigen in the surface epithelium and subepithelial macrophages, whereas the crypt epithelium was unaffected. In the third trial the pigs were sacrificed 5 weeks after inoculation, 1 week later than in trials 1 and 2. At this time, the number of intestinal sections with PE, i.e. L. intracellularis infection of immature crypt epithelial cells, was limited, but immune staining with the specific antibody often revealed antigen within macrophages surrounding normally appearing crypts. Thus, as the crypt morphology was restored we believe that the findings of antigen positive surface enterocytes and subepithelial macrophages indicate diminishing of the infection, corresponding to the general decrease in PCR positive fecal samples found at these time points. Presence of L. intracellularis antigen between crypts is in a previous study suggested to be the result of recovery after infection (Jensen et al., 1997). Late stage faecal excretion of L. intracellularis and presence of bacteria in apical cytoplasm of immature crypt enterocytes in N-INF pig indicate that the infections in the control groups were initiated at the termination of the investigation. We believe that the infections may be a cross contamination of bacteria from experimentally infected groups possibly transferred by attendants from one pen to the other. As the infection appears late in the investigation it did not influenced the overall evaluation of growth performance and faecal excretion data. In conclusion, we have established an infection model that yields high morbidity inducing reduced growth performance as wells as typical lesions of PE in accordance with the current status of L. intracellularis infection in Denmark. Furthermore, we have demonstrated that fermented liquid feed delayed the excretion of L. intracellularis and that lactic acid to some degree reduce the lesions, whereas a coarse ground and non-pelleted diet had no effect on the infection. The presented data underline the importance of evaluating feeding strategies as alternative methods to control L. intracellularis.
Acknowledgements The technical assistance of E.K.A. Jensen, M. Rødkjær, U.L. Andreasen, A.R. Pedersen, L.T.M. Nguyen and S.H. Christensen is gratefully appre-
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ciated. Part of this work was supported by the Research Secretary of the Ministry of Food, Agriculture and Fisheries (Project number Alt-98-1). References Boesen, H.T., Jensen, T.K., Jungersen, G., Riber, U., Boye, M., Møller, K., 2004. Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen. Vet. Microbiol. (in review). Brunsgaard, G., 1998. Effects of cereal type feed particle size on morphological characteristics, epithelial cell proliferation and lectin binding patterns in the large intestine of pigs. J. Anim. Sci. 76, 2787–2798. Canibe, N., Jensen, B.B., 2001. Effect of feed structure and processing and addition of formic acid to the feed on microbial ecology of the gastrointestinal tract of pigs,AFCA Workshop, Oslo, Norway. Guedes, R.M.C., Gebhart, C.J., 2003. Comparison of intestinal mucosa homogenate and pure culture of the homologous Lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine. Vet. Microbiol. 93, 159–166. Guedes, R.M.C., Gebhart, C.J., Winkelman, N.L., Mackie-Nuss, R.A., 2002. A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy. J. Vet. Diagn. Invest. 14, 420–423. Højberg, O., Canibe, N., Knudsen, B., Jensen, B.B., 2003. Potential rates of fermentation in digesta from the gastrointestinal tract of pigs: effect of feeding fermented liquid feed. Appl. Environ. Microbiol. 69, 408–418. Jensen, B.B., Højberg, O., Mikkelsen, L.L., Hedemann, M.S., Canibe, N., 2003a. Enhancing intestinal function to treat and prevent intestinal disease. In: Proceedings of the International Symposium of Digestive Physiology in Pigs, vol. 1, Banff, AB, Canada, pp. 103–119. Jensen, T.K, Christensen, B.B., Boye, M., 2003b. Lawsonia intracellularis infection in the large intestines of pigs. APMIS, March 2004. Jensen, T.K., Møller, K., Leser, T.D., Jorsal, S.E., 1997. Comparison of histology, immunohistochemistry and polymerase chain reaction for detection of Lawsonia intracellularis in natural porcine proliferative enteropathy. Eur. J. Vet. Pathol. 3, 115–123. Johansen, M., Bækbo, P., Jensen, T.K., Møller, K., Nielsen, V.R., 2002. Attempt to eradicate Lawsonia intracellularis by medication in 9 sow herds; preliminary results. In: Proceedings of the 17th International Pig Veterinary Society Congress, vol. 1, p. 222. Lawson, G.H.K., Gebhart, C.J., 2000. Proliferative enteropathy. J. Comp. Pathol. 122, 77–100.
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Leser, T.D., Lindecrona, R.H., Jensen, T.K., Jensen, B.B., Møller, K., 2000. Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl. Environ. Microbiol. 66, 3290–3296. Lindecrona, R.H., Jensen, T.K., Jensen, B.B., Leser, T.D., Jiufeng, W., Møller, K., 2003a. The influence of diet on the development of swine dysentery upon experimental infection. Anim. Sci. 76, 81–87. Lindecrona, R.H., Jensen, T.K., Møller, K., 2003b. Feeding study with pigs experimentally infected with B. pilosicoli. Vet. Rec., June 2003. Lindecrona, R.H., Jensen, T.K., Andersen, P.H., Møller, K., 2002. Application of a 50 nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs. J. Clin. Microbiol. 40, 984–987. Lomax, L.G., Glock, R.D., Hogan, J.E., 1982. Experimentally induced porcine proliferative enteritis in specific-pathogen-free pigs. Am. J. Vet. Res. 43, 1615–1621. Mapother, M.E., Joens, L.A., Glock, R.D., 1987. Experimental reproduction of porcine proliferative enteritis. Vet. Rec. 121, 533–536. Maribo, H., Olsen, L.E., Jensen, B.B., Miquel, N., 2000. Different doses of organic acids to piglets. Publication no. 460. The National Committee for Pig Production, Copenhagen, Denmark (in Danish). McOrist, S., Jasni, S., Mackie, R.A., MacIntyre, N., Neef, N., Lawson, G.H.K., 1993. Reproduction of porcine proliferative enteropathy with pure cultures of ileal symbiont intracellularis. Infect. Immun. 61, 4286–4292. McOrist, S., Mackie, R.A., Lawson, G.H.K., 1995. Antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy. J. Clin. Microbiol. 33, 1314– 1317. Mikkelsen, L.L., Jensen, B.B., 1998. Performance and microbial activity in the gastrointestinal tract of piglets fed fermented liquid feed at weaning. J. Anim. Feed Sci. 7, 211–215. Siba, P.M., Pethick, D.W., Hampson, D.J., 1996. Pigs experimentally infected with Serpulina hyodysenteriae can be protected from developing swine dysentery by feeding them a highly digestible diet. Epidemiol. Infect. 116, 207–216. Smith, S.H., McOrist, S., Green, L.E., 1998. Questionnaire survey of proliferative enteropathy on British pig farms. Vet. Rec. 142, 690–693. Stege, H., Jensen, T.K., Møller, K., Bækbo, P., Jorsal, S.E., 2000. Prevalence of intestinal pathogens in Danish finishing pig herds. Prev. Vet. Med. 46, 279–292. Stege, H., Jensen, T.K., Møller, K., Bækbo, P., Jorsal, S.E., 2001. Risk factors for intestinal pathogens in Danish finishing pig herds. Prev. Vet. Med. 50, 153–164. WHO Report, 2003. Impacts of antimicrobial growth promoter termination in Denmark.
The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge Henriette T. Boesena,*, Tim K. Jensena, Anja S. Schmidta, Bent B. Jensenb, Søren M. Jensena, Kristian Møllera a
Danish Institute for Food and Veterinary Research, Bu¨lowsvej 27, DK-1790 Copenhagen V, Denmark b Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark Received 26 November 2003; received in revised form 17 May 2004; accepted 14 June 2004
Abstract The objective of this investigation was to study if different feeding strategies influence experimental infections of pigs with Lawsonia intracellularis, the causative agent of proliferative enteropathy. In three sequential trials, a total of 144 weaned pigs were fed five different diets all made from a standard diet based on wheat and barley as carbohydrate source and soybean as protein source. The five diets were: a standard diet (fine ground and pelleted), the standard diet fed as fermented liquid feed, the standard diet added 1.8% formic acid, the standard diet added 2.4% lactic acid and a diet similar to the standard diet (made from the same ingredients), but fed coarse ground. Twenty-four pigs on each diet were orally inoculated with L. intracellularis and growth performance and faecal excretion of bacteria were monitored. Twenty-four pigs fed the standard diet were included as not experimentally infected controls. Pigs in the first two trials were sacrificed 4 weeks post-inoculation, whereas animals in the third trial were sacrificed after 5 weeks. Pigs in all experimentally infected groups excreted L. intracellularis. The fermented liquid diet delayed the excretion of L. intracellularis and furthermore, pigs fed the standard diet supplemented with lactic acid had limited pathological lesions when the intestines were examined 4 weeks after inoculation. The growth performance was reduced in pigs experimentally challenged with L. intracellularis, however the prevalence and severity of diarrhea was limited. # 2004 Elsevier B.V. All rights reserved. Keywords: Lawsonia intracellularis; Proliferative enteropathy; Feed; Experimental infection; Pigs
1. Introduction Lawsonia intracellularis is an obligate intracellular bacterium causing proliferative enteropathy (PE). The * Corresponding author. Tel.: +45 72 34 60 00; fax: +45 72 34 60 01. E-mail address: [email protected] (H.T. Boesen).
infection causes diarrhea, retarded growth and/or sudden death in pigs and is one of the most economically important diseases in the swine industry worldwide (Lawson and Gebhart, 2000). In a recent Danish study L. intracellularis was detected by PCR in 93.7% of 79 randomly selected herds (Stege et al., 2000). The disease is characterized by adenomatous proliferation of epithelial cells infected by the bacterium and is
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.06.008
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grossly seen as a thickening of the intestinal mucosa in the aboral part of the small intestines, particularly the ileum, but may involve cecum and colon as well (Lawson and Gebhart, 2000). Antimicrobial growth promoters (AGP) has been used in many countries to control subclinical infections with L. intracellularis, however in Denmark the use of AGP was terminated in 1999 and since then the use of therapeutic antibiotic for pigs has increased, especially antibiotics effective of controlling L. intracellularis infections (WHO Report, 2003). Alternative methods to control the intestinal pathogen are therefore relevant to investigate. Diet fed to healthy animals is an important factor, which can regulate the microbial flora of the intestines (Leser et al., 2000). Different diets have been shown to affect the intestinal function in pigs by several mechanisms. Recently it has been shown, that feeding pigs a diet consisting of cooked rice and animal protein reduces the severity of the experimental infection with Brachyspira pilosicoli (Lindecrona et al., 2003b) and B. hyodysenteriae (Siba et al., 1996). Fermented liquid feed decreases the bacterial population throughout the gastrointestinal tract of pigs (Mikkelsen and Jensen, 1998; Højberg et al., 2003), and it is demonstrated to have a preventing effect on the development of swine dysentery (Lindecrona et al., 2003a). Coarse ground non-pelleted feed affects intestinal morphology in terms of an increase in height and volume of crypts (Brunsgaard, 1998) and has in a previous study been shown to reduce the prevalence of L. intracellularis (Stege et al., 2001). Both fermented liquid feed, organic acid and coarse non-pelleted feed increase the concentration of organic acids and lower the pH in the stomach. Consequently, enteric bacteria like Salmonella spp. and Escherichia coli are killed or reduced in number before reaching the parts of the gastrointestinal tract in which they will normally proliferate (Jensen et al., 2003a). Lactic acid and formic acid have different effects on the gastrointestinal microbiota. Lactic acid tends to increase the number of lactic acid bacteria, increase the population of yeast and to decrease the number of coliform bacteria along the gastrointestinal tract (Maribo et al., 2000). Formic acid, on the other hand, decreases the number of lactic acid bacteria, coliform bacteria and yeast along the gastrointestinal tract (Maribo et al., 2000; Canibe and Jensen, 2001).
In the current study, the effects of fermenting, acidifying and grinding the feed on L. intracellularis colonization and the development of PE after experimental challenge with L. intracellularis were investigated. Important evaluation parameters were growth performance and faecal excretion of bacteria, whereas induction of diarrhea was regarded a less critical parameter as many L. intracellularis infected herds in Denmark only have a history of unthriftiness among the pigs.
2. Material and methods 2.1. Animals and treatment groups A total of 144 (three trials of 48) pigs (Danish Landrace/Yorkshire/Duroc) of mixed sex, 6–7 weeks of age and weighing 10.4 2.1 kg (mean S.D.) (range 6.4–15.5 kg) were purchased from two high health herds known to be free of B. hyodysenteriae and L. intracellularis after medicated eradication program (Johansen et al., 2002). The absence of L. intracellularis were confirmed by PCR analysis (Lindecrona et al., 2002) of 50 faeces samples from pigs at different age group collected from the herds twice (with 2 weeks interval) prior to the experiments. One week before the end of the experiments faecal samples were tested for the presence of Salmonella spp. and pathogenic Brachyspira spp. by bacteriological culture (Stege et al., 2000). Seven pigs (four pigs in trial 2 and three pigs in trial 3) died or were euthanized due to illness not related to L. intracellularis during the study. 2.2. Experimental design The pigs were allocated to six separate pens and each pen contained eight pigs in each sequential trial. Five groups of pigs were offered one of five diets and each group was infected with L. intracellularis after 1 week on the diet. The sixth pen housed groups of eight control animals not exposed to L. intracellularis (not experimentally infected, N-INF). The pens were separated from each other by ‘‘open-mesh wire’’ walls, which permitted some contact between the animals. The pens had slatted floors and no bedding was provided. The N-INF group was isolated from the infected groups
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by a gangway and attendants always first sampled from the N-INF pigs before sampling from the infected groups. The whole stable with pens was formalin sterilized prior to introduction the pigs and no other pigs were kept within the premises during the experimentation. All procedures of animal handling and experimentation were performed under veterinary supervision and according to recommendations by the Danish Committee of Animal Experimentation.
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by grinding through a 5 mm sieve in the same mill. The fermented liquid feed was prepared in an 80-L closed tank with continuous stirring as described by Mikkelsen and Jensen (1998). In short, the standard diet was mixed with water at a ratio of 1:2.5 (w/w). The feed was allowed to pre-ferment at 24 8C for 3–4 days before onset of the experiment. At each feeding, one-quarter of the content was taken from the tank, and the tank was refilled with an equivalent amount of new food and water. The pigs were fed the experimental diets ad libitum.
2.3. Diets 2.4. Experimental challenge Five different diets all made from a standard diet based on wheat and barley as carbohydrate source and soybean as protein source (Table 1) were used in the present experiment. The five diets were: STD, standard diet (fine ground and pelleted); FLF, the standard diet fed as fermented liquid feed; FAC, the standard diet added 1.8% formic acid; LAC, the standard diet added 2.4% lactic acid and COARSE, a diet similar to the standard diet (made from the same ingredients, Table 1) but fed coarse ground. The NINF groups were fed STD. The fine pelleted diets (STD, FLF, FAC and LAC) were ground through a 2 mm sieve while the coarse ground diet was obtained
Table 1 Composition of the diet Item
Feed (g/kg)
Wheat Barley Dehulled toasted soybean meal Rapeseed meal Sunflower meal Animal fat Calcium carbonate Dicalcium phosphate Sodium chloride L-Lysine, 40% Vitamin and mineral premixa DL-Methionine, 40% Threonine, 50%
360.8 360.7 122.9 80.0 20.0 25.3 8.0 11.5 3.9 2.9 2.0 1.2 0.8
a
Supplied per kilogram of diet: 2200 IU Vitamin A; 500 IU Vitamin D3; 30,000 mg Vitamin E; 1100 mg Vitamin K3; 1100 mg Vitamin B1; 2000 mg of Vitamin B2; 1650 mg of Vitamin B6; 5500 mg D-pantothenic acid; 11000 mg niacin; 27.5 mg biotin; 11 mg Vitamin B12; 25,000 mg FeSO4x7H2O; 40,000 mg ZnO; 13,860 mg MnO; 10,000 mg CuSO4x5H2O; 99 mg KI; 150 mg Na2SeO3.
PE-affected small intestines collected from two farms were utilized for the preparation of the L. intracellular homogenate inoculum. Frozen (80 8C) intestines were thawed and the mucosa scraped using a sterile scalpel. The scraped mucosa was combined with sucrose–potassium–glutamate (SPG, pH 7.0) and homogenized in a blender for 2 min. The inoculum was kept at 4 8C until use on the same day. L. intracellularis was quantified in the homogenate by making serial 1:10 dilutions of the homogenate in SPG with 5% fecal calf serum. Six-well glass slides were coated with 10 ml of each dilution, dried at room temperature, fixed in methanol, and stained by an indirect immunofluorescence method (Jensen et al., 1997) using a monoclonal antibody specific for L. intracellularis (Law1-DK) (Boesen et al., 2004). The number of fluorescent bacteria was counted in 10 view fields at 40 objective representing 1/25 of the total well. Pigs were fasted for 24 h before inoculation. They were infected via stomach tube by administering approximately 25 ml of L. intracellularis homogenate containing 3.6 1.2 108 bacteria/ml (mean S.D.). The N-INF groups received 25 ml SPG. 2.5. Monitoring of the infection Faecal samples were collected from each pig 24 h prior to the L. intracellularis inoculation and then every 2–3 days during the study. Samples were analyzed by real time PCR for detection of L. intracellularis DNA (Lindecrona et al., 2002). When L. intracellularis was detected from the same pig in two consecutive faecal samples, the pig was con-
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sidered to be excreting L. intracellularis in the period between these two samples. The pigs were weighed every 7 days throughout the experimental period to determine the growth rate. The pigs were observed daily for clinical signs of PE, in particular diarrhea. At the end of the experimentation (4 or 5 weeks (trial 3) after challenge) the animals were sacrificed by means of captive bolt pistol followed by pitching and exsanguination. The animals were necropsied and tissue samples were taken from ileum, caecum and top of the spiral colon for histopathologically examination. The samples were placed in 10% neutralbuffered formalin. After fixation the material was dehydrated, embedded in paraffin, sectioned at 3 mm and immune stained with a L. intracellularis specific monoclonal antibody (Law1-DK) according to Jensen et al. (2003b). Briefly, after deparaffinization in xylene the sections were rehydrated and treated with 0.6% H2O2 in Trisma base buffer (TBS) (50 mM Tris, 150 mM NaCl, pH 7.6) for 20 min. Then washed 3 min 5 min in TBS followed by incubation in a moisture chamber for 30 min with the MAb in TBS. Washed 3 min 5 min in TBS before incubation with EnVision+TM (DAKO, Glostrup, Denmark) for 30 min. After 3 min 5 min wash in TBS the reaction was developed for 15–20 min with a solution of 3-amino-9-ethylcarbozole (KemEnTec, Copenhagen, Denmark), counterstained with Mayer’s haematoxylin and mounted with Glycergel (DAKO). The immune stained sections were examined for the presence of L. intracellularis antigen in the crypt
and surface epithelium of the intestines as well as within subepithelial macrophages. The animals were scored according to the severity and extension of the lesions: (+) focal detection of L. intracellularis antigen within macrophages only, (++) focal detection of L. intracellularis within crypt epithelium (focal PE), (+++) multifocal detection of L. intracellularis infected crypts (PE), (++++) detection of L. intracellularis infected crypts diffusely within the mucosa (PE). Only pigs surviving during the whole experimental period were included in the weight, faecal excretion and immunohistochemistry data set. Table 2 includes the number of pigs included in the data material.
2.6. Data analysis Data were statistically analyzed by one-way analysis of variance (ANOVA) followed either by Dunnett’s multiple comparison test for comparing mean duration of faecal excretion between pigs feed STD and pigs feed the modified diets or by Tukey’s multiple range test for comparison of average daily weight gain. Results are expressed as mean standard error of the mean. Data analyses were performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA, USA. Power analyses and effect size index were carried out using G*power, http://www.psycho.uni-duesseldorf. de/aap/projects/gpower/index.html.
Table 2 Faecal excretion of L. intracellularis after experimental challenge and actual number of inoculated pigs given different diets in triplicate trials Dieta
Trial 1/2/3 Number of pigs inoculated or not experimental infectedb
Excretion first detected after day 5 post-exposure (days after inoculation)
Pigs that excrete L. intracellularis (no. of pigs)
STD FLF FAC LAC COARSE
8/8/8 8/8/7 8/8/8 8/7/8 8/5/7
8/8/8 10/10/10 8/6/8 6/8/10 8/6/8
7/8/4 8/7/7 8/7/8 7/2/7 8/3/7
N-INF
8/8/7
15/27/0
5/3/0
a
STD: fine ground and pelleted standard diet; FLF: fermented liquid feed; FAC: standard diet with formic acid; LAC: standard diet with lactic acid; COARSE: coarse ground standard diet; N-INF: not experimental infected pigs fed the STD diet. b Pigs inoculated with L. intracellularis or not experimental infected, which survived through the entire experiment period. Numbers represent sequential trial results, e.g. 8/7/8 represents 8, 7 and 8 animals in sequential trials.
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3. Results 3.1. Faecal excretion of L. intracellularis following experimental challenge The percentages of pigs that excrete L. intracellularis in the different diet groups are presented in Fig. 1. Two to three weeks after inoculation 65–90% of the pigs from all diet groups excreted L. intracellularis. Four weeks after inoculation 40–65% of the pigs still excreted L. intracellularis. Besides excretion of L. intracellularis in faeces immediately after inoculation, the excretion of bacteria was not detectable until 10 days after inoculation in the group fed the FLF (Fig. 1 and Table 2). In all other diet groups the excretion was initiated already 6–8 days after inoculation (except for diet group LAC in trial 3 which initiated excretion after 10 days). In Table 2 the number of pigs experimentally inoculated with L. intracellularis, which survived throughout the study are listed for each trial together with the number of pigs excreting L. intracellularis. Of the experimentally infected pigs, 14% did not excrete L. intracellularis at any time throughout the study. Eight pigs from the N-INF groups excreted L. intracellularis at the termination of the observation period (trials 1 and 2, Fig. 1 and Table 2). The mean duration of faecal excretion in pigs that
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excreted L. intracellularis (excluding data immediately post-inoculation) is illustrated in Fig. 2. In the different diet groups the mean duration of faecal excretion varied from 11.7 to 15.6 days. The mean duration of faecal excretion in the FLF fed group was significantly lower than in pigs fed the STD (P < 0.05). No significant difference in duration of bacteria excretion was observed between the STD group and any of the other diet groups. The power of the test was 67% with an effect size index of 0.31. Calculations were based on an accumulation of data from the three trials, as there was a good accordance in faecal excretion between the different trials. 3.2. Growth rate Pigs were weighed throughout the experimental period and the average weight for pigs on the different diets are presented in Fig. 3. For the first 2 weeks after inoculation the average weight gain was 215 86 g/ day (mean S.D.) for pigs in all the tested diet groups. Differences in growth rate between the L. intracellularis experimentally infected diet groups and the N-INF group initiated 2 weeks after inoculation, and the average daily weight gain from that time point to sacrifice is illustrated in Fig. 4. In an interval of 2–4 weeks after the time of inoculation the
Fig. 1. The faecal excretion after experimental challenge with L. intracellularis in pigs offered various diets. Values are total number of pigs excreting L. intracellularis in three trials in percentage of the total number of pigs inoculated. The pigs were fed either STD, n = 24 (~); FLF, n = 23 (&); FAC, n = 24 (~); LAC, n = 23 (); or COARSE, n = 20 (^). Not experimentally infected pigs were fed the STD diet, n = 23 (*).
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Fig. 2. The duration of faecal excretion after experimental challenge with L. intracellularis in pigs offered various diets. Values are mean excretion days + S.E.M. of pigs that at any time of the experiment excreted L. intracellularis. Groups with (*) differ from the STD group at P < 0.05 (by one-way analysis of variance with Dunnett’s adjustment).
average daily weight gain of the N-INF group was superior compared to the individual experimentally infected groups in the same time interval. This difference was, however, only significant (P < 0.01) when comparing the experimentally infected pigs feed the STD or FLF with the N-INF group. The five diet groups experimentally infected with L. intracellularis showed no difference in terms of weight gain. The power of the study was 90% with an effect size index of 0.36. Calculations were based on an accumulation
of data from the three trials, as there was a good accordance in weight gain between the different trials. 3.3. Clinical outcome The clinical signs of L. intracellularis infection in terms of diarrhea were very limited, even in pigs excreting bacteria for longer periods. Except from the pigs with E. coli diarrhea (see Section 3.4) a few pigs from each group had signs of loose faeces,
Fig. 3. The average weight of pigs offered various diets and experimentally challenged with L. intracellularis. The pigs were fed either STD, n = 24 (~); FLF, n = 23 (&); FAC, n = 24 (~); LAC, n = 23 (); or COARSE, n = 20 (^). One group of pigs was fed the STD and left not experimental infected; N-INF, n = 23 (&). Values are mean weight S.E.M.
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Fig. 4. The average daily weight gain from 2 to 4 weeks after experimental challenge with L. intracellularis in pigs offered various diets. The results are mean weight gain + S.E.M. Groups sharing same letters are significant different from each other at P < 0.01 (by one-way analysis of variance with Tukey’s adjustment).
but we seldom observed diarrhea (watery and grey faces). 3.4. Other infections Three weeks after inoculation with L. intracellularis faecal samples were tested negative for the presence of B. pilosicoli, B. hyodysenteriae and Salmonella spp. by bacteriological culturing. In the second and third trial, some pigs suffered from E. coli associated diarrhea in the second week of experimentation. As especially pigs from diet groups fed LAC and COARSE in trial 2 were affected both groups were treated twice i.m. with 225 mg amoxicillin (Clamoxyl1). Seven pigs (four pigs in trial 2 and three pigs in trial 3) died or were euthanised due to the E. coli infection. 3.5. Necropsy and histopathology Grossly thickening of intestinal mucosa in the aboral part of the small intestine was observed 4 weeks after inoculation in 13–38% of the pigs fed the STD, FLF, FAC and COARSE, but not in pigs fed the LAC. In Fig. 5 an ileum with typically thickened mucosa is compared to ileum from a N-INF pig. In trial 3, where the pigs were kept alive for an extra week, a similar grossly thickening of the intestinal
mucosa was only seen in one and two pigs from diet groups LAC and COARSE, respectively (data not shown). Large intestinal gross lesions were not observed in any pigs. In trials 1 and 2, a few pigs in all diet groups showed ulceration of pars oesophagea of the stomach. Extragastro-intestinale lesions were not observed. Diffuse PE with L. intracellularis in the apical cytoplasm of immature crypt enterocytes was demonstrated in the most severely affected ilea together with superficial necrosis. In the ileum from some pigs without gross lesions, L. intracellularis antigen was detected within the apical cytoplasm of surface enterocytes as well as in subepithelial macrophages between normally appearing crypts (Fig. 6). The extent of L. intracellularis infection in the large intestines was only minor compared to the lesions in ileum (data not shown). Fig. 7 illustrates the scoring of positive immune staining for L. intracellularis in ileal sections of pigs inoculated with the bacterium and sacrificed 4 or 5 weeks post-exposure. The degree of infection at 4 weeks after inoculation (trials 1 and 2) tended to be less severe in the diet group fed LAC, as L. intracellular antigen was detected within subepithelial macrophages only. Diet groups STD, FLF, FAC and COARSE all included pigs with proliferative immature enterocytes infected by L. intracellularis in the crypts at the time of sacrifice (Fig. 7a and b). Five
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Fig. 5. Gross appearance of an ileum with diffuse PE from a pig challenged with L. intracellularis (COARSE) (top) compared to the ileum from a N-INF control (bottom). The mucosa of the affected ileum is evidently thickened and ridged.
Fig. 6. Ileum from a pig sacrificed 4 weeks after experimental challenged with L. intracellularis. Immunohistochemical detection of L. intracellularis antigen (brownish-red) diffusely within large, subepithelial macrophages and as small rods in the apical cytoplasm of surface enterocytes (inserted). The adjacent crypts are lined by mature enterocytes including goblet cells.
weeks after inoculation (trial 3), fewer pigs showed PE with infected immature enterocytes, but L. intracellularis antigen was demonstrated within subepithelial macrophages (Fig. 7c). In seven of the eight N-INF pigs from trials 1 and 2, which excreted L. intracellularis during the experiment, bacteria were detected in proliferative immature enterocytes. Furthermore, L. intracellularis were present in sections of ileum of four N-INF pigs from trial 3, although the pigs did not excrete the bacterium (results not shown).
4. Discussion This study examined the influence of different diets on the colonization of L. intracellularis in weaned pigs after an experimental challenge. Diet type and feeding systems have not been identified as risk factors in a questionnairy survey on British pig farms (Smith et al., 1998). However, this type of investigation may have limitations as to exact knowledge of the different management parameters, content of feed and exact diagnosis of pigs, and we therefore considered it
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Fig. 7. Scoring of positive immune staining for L. intracellularis in ileal sections after experimental challenge in pigs offered various diets. Values are total numbers of pigs found positive by immunohistochemistry in percentage of the total number of pigs inoculated. Pigs were either sacrificed 4 weeks ((a) trial 1; and (b) trial 2) or 5 weeks ((c) trial 3) after inoculation. Scoring: (+) focal detection of L. intracellularis antigen within macrophages only, (++) detection of L. intracellularis focally within enterocytes (focal PE), (+++) detection of L. intracelularis multifocally within enterocytes, PE, (++++) detection of L. intracellularis diffusely within enterocytes, PE.
relevant to study the influence of diet on L. intracellularis colonization in experimentally infected pigs. The mean duration of faecal excretion of L. intracellularis was significantly lower in pigs consuming a fermented liquid standard diet compared to pigs
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fed a non-fermented diet. In previous experimentally infection studies with two other intestinal pathogens, B. hyodysenteriae and B. pilosicoli, the effect of similar diet groups were tested (Lindecrona et al., 2003a,b). In these studies there were a preventing effect of the fermented diet on the B. hyodysenteriae infection, but no effect on experimental infection with B. pilosicoli. Feeding fermented liquid feed reduces pH to between 4 and 5 in the stomach and the density of coliform bacteria is decreased throughout the gastrointestinal tract in pigs (Mikkelsen and Jensen, 1998). The acidity of the feed might impair the survival of L. intracellularis and explain the reduced time of L. intracellularis excretion in this diet group. Additionally, fermented liquid feed contains high number of lactobacilli and high concentration of lactic acid, and feeding FLF to pigs leads to higher number of lactobacilli in the small intestine (Mikkelsen and Jensen, 1998), which may delay the colonization of L. intracellularis. The addition of organic acids to the feed, either as lactic acid or as formic acid, had no reducing effect on the mean duration of faecal excretion of L. intracellularis, however, as the study only had 67% power to find a statistically significant difference, we can not necessarily conclude that the treatment was ineffective. To reach a firm conclusion on the absence of effect the sample size will have to be increased. In the present investigation, the pigs fed the diet with lactic acid had fewer lesions in the intestine 4 weeks after inoculation compared to the pigs fed the STD. Pigs fed both diets supplemented with organic acids had a slightly improvement of growth rate compared to the pigs fed the STD. The use of lactic acid is known to increase the concentration of lactic acid and to decrease pH in stomach content of pigs and consequently to kill coliform bacteria like E. coli and Salmonella spp. (Jensen et al., 2003a). A similar effect of the LAC on the gastrointestinal ecosystem may also explain the reduction of the infection with L. intracellularis. It has previously been reported that home-mixed diet is associated with reduced prevalence of L. intracellularis (Stege et al., 2001). In the present study, feeding coarse ground, non-pelleted feed, which mimicked a home-mixed diet, did not reduce the infection with L. intracellularis, compared with the fine-ground and pelleted standard feed. Differences in observations may be a result of the setup of the investigations. In the present study, the infections were experimental whereas the study by Stege and
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coworkers was based on a questionnaire where natural infections of L. intracellularis in 79 randomly selected herds were investigated. In the second trial of the present investigation, two diet groups of pigs (LAC and COARSE) suffered from an outbreak of E. coli associated diarrhea shortly after the L. intracellularis inoculation, which compelled us to treat pigs in these groups with amoxicillin. This antibiotic may have had an effect on L. intracellularis either directly as L. intracellularis is inhibited by the related ampicillin (McOrist et al., 1995) or indirectly by an effect on the general bacterial community in the intestine, which may affect the degree of the infection. L. intracellularis has previously been shown to be dependent on the presence of other enterobacteria, as an infection could not be experimentally established in gnotobiotic pigs (McOrist et al., 1993). These facts may explain the low infection rate in the amoxicillintreated groups, however exclusion of data obtained in these two diet groups does not change the statistical assessment of the presented influence of diet on L. intracellularis infections. The in vitro maintenance of L. intracellularis is difficult, and we therefore conducted our infection model using infected intestinal mucosa from PE-affected pigs. PE has successfully been experimentally reproduced in pigs inoculated with infected intestinal mucosa in several studies (Lomax et al., 1982; Mapother et al., 1987; Guedes and Gebhart, 2003), and our experimental model resulted in mild to moderate clinical symptoms. Pigs excreted L. intracellularis starting 1 week postinoculation, but the infection was not associated with mortality as in the infection model established by Guedes and coworkers (Guedes et al., 2002). The differences in morbidity of experimental infection models may be the result of different pathogenecity of the bacteria used for challenge. However, it is noticeable that all infected groups in the present study had a lower average weight gain than the N-INF group. All together, the present infection model yielded a morbidity and severity, which is in accordance with the current Danish field situation of the disease. Furthermore, lesions found in several of the pigs sacrificed 4 weeks post-challenge showed evident PE, similar to that described in natural as well as in experimental infection (Jensen et al., 1997; McOrist et al., 1993). When the intestines were histopathologically examined some pigs revealed only L. intracellularis
antigen in the surface epithelium and subepithelial macrophages, whereas the crypt epithelium was unaffected. In the third trial the pigs were sacrificed 5 weeks after inoculation, 1 week later than in trials 1 and 2. At this time, the number of intestinal sections with PE, i.e. L. intracellularis infection of immature crypt epithelial cells, was limited, but immune staining with the specific antibody often revealed antigen within macrophages surrounding normally appearing crypts. Thus, as the crypt morphology was restored we believe that the findings of antigen positive surface enterocytes and subepithelial macrophages indicate diminishing of the infection, corresponding to the general decrease in PCR positive fecal samples found at these time points. Presence of L. intracellularis antigen between crypts is in a previous study suggested to be the result of recovery after infection (Jensen et al., 1997). Late stage faecal excretion of L. intracellularis and presence of bacteria in apical cytoplasm of immature crypt enterocytes in N-INF pig indicate that the infections in the control groups were initiated at the termination of the investigation. We believe that the infections may be a cross contamination of bacteria from experimentally infected groups possibly transferred by attendants from one pen to the other. As the infection appears late in the investigation it did not influenced the overall evaluation of growth performance and faecal excretion data. In conclusion, we have established an infection model that yields high morbidity inducing reduced growth performance as wells as typical lesions of PE in accordance with the current status of L. intracellularis infection in Denmark. Furthermore, we have demonstrated that fermented liquid feed delayed the excretion of L. intracellularis and that lactic acid to some degree reduce the lesions, whereas a coarse ground and non-pelleted diet had no effect on the infection. The presented data underline the importance of evaluating feeding strategies as alternative methods to control L. intracellularis.
Acknowledgements The technical assistance of E.K.A. Jensen, M. Rødkjær, U.L. Andreasen, A.R. Pedersen, L.T.M. Nguyen and S.H. Christensen is gratefully appre-
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ciated. Part of this work was supported by the Research Secretary of the Ministry of Food, Agriculture and Fisheries (Project number Alt-98-1). References Boesen, H.T., Jensen, T.K., Jungersen, G., Riber, U., Boye, M., Møller, K., 2004. Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen. Vet. Microbiol. (in review). Brunsgaard, G., 1998. Effects of cereal type feed particle size on morphological characteristics, epithelial cell proliferation and lectin binding patterns in the large intestine of pigs. J. Anim. Sci. 76, 2787–2798. Canibe, N., Jensen, B.B., 2001. Effect of feed structure and processing and addition of formic acid to the feed on microbial ecology of the gastrointestinal tract of pigs,AFCA Workshop, Oslo, Norway. Guedes, R.M.C., Gebhart, C.J., 2003. Comparison of intestinal mucosa homogenate and pure culture of the homologous Lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine. Vet. Microbiol. 93, 159–166. Guedes, R.M.C., Gebhart, C.J., Winkelman, N.L., Mackie-Nuss, R.A., 2002. A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy. J. Vet. Diagn. Invest. 14, 420–423. Højberg, O., Canibe, N., Knudsen, B., Jensen, B.B., 2003. Potential rates of fermentation in digesta from the gastrointestinal tract of pigs: effect of feeding fermented liquid feed. Appl. Environ. Microbiol. 69, 408–418. Jensen, B.B., Højberg, O., Mikkelsen, L.L., Hedemann, M.S., Canibe, N., 2003a. Enhancing intestinal function to treat and prevent intestinal disease. In: Proceedings of the International Symposium of Digestive Physiology in Pigs, vol. 1, Banff, AB, Canada, pp. 103–119. Jensen, T.K, Christensen, B.B., Boye, M., 2003b. Lawsonia intracellularis infection in the large intestines of pigs. APMIS, March 2004. Jensen, T.K., Møller, K., Leser, T.D., Jorsal, S.E., 1997. Comparison of histology, immunohistochemistry and polymerase chain reaction for detection of Lawsonia intracellularis in natural porcine proliferative enteropathy. Eur. J. Vet. Pathol. 3, 115–123. Johansen, M., Bækbo, P., Jensen, T.K., Møller, K., Nielsen, V.R., 2002. Attempt to eradicate Lawsonia intracellularis by medication in 9 sow herds; preliminary results. In: Proceedings of the 17th International Pig Veterinary Society Congress, vol. 1, p. 222. Lawson, G.H.K., Gebhart, C.J., 2000. Proliferative enteropathy. J. Comp. Pathol. 122, 77–100.
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