Sponsors University of Minnesota College of Veterinary Medicine College of Agricultural, Food and Environmental Sciences Extension Service Swine Center Editors w. Christopher Scruton Stephen Claas Layout David Brown LogoDesign Ruth Cronje, and Jan Swanson; based on the original design by Dr. Robert Dunlop Cover Design Sarah Summerbell ii The University of Minnesota is committed to the policy that all persons shall have equal access to its programs, facilities, and employment without regard to race, color, creed, religion, national origin, sex, age, marital status, disability, public assistance status, or sexual orientation. 2001 Allen D. Leman Swine Conference Immunity to Lawsonia intracellularis A. M. Collins B.Ag.Sc., PhD; M. van Dijk; N. Q. Vu D.V.M., M.Sc.; 1. Pozo D.V.M.; and R. 1. Love B.V.Sc., PhD Faculty of Veterinary Science, University of Sydney, Australia. The porcine proliferative enteropathies (PPE) are a group of conditions that affect pigs of all ages housed in a range of management systems worldwide. The chronic form of PPE, commonly known as 'ileitis,' may lead to clinical signs including diarrhea and reduced weight gain. Prolif- erative hemorrhagic enteropathy (PHE), the acute form of disease, is characterized by bloody diarrhea caused by a massive hemorrhage from the ileal mucosa. The etio- logic agent of the porcine proliferative enteropathies is Lawsonia intracellularis, an obligate intracellular bacterium. PPE is currently controlled by medicating pigs in-feed or in-water using a range of antibioticsl,2,3,4·5. Some current medication strategies recommend preventing L. intracellularis infection by continuous medication with high levels of antibiotics, which may simply extend the period of susceptibility to L.intracellularis, delaying in- fection until the removal of antibiotics. This becomes a problem when susceptible finisher and breeding pigs be- come infected with L.intracellularis. Finisher pigs in- fected with L.intracellularis experience more severe clini- cal signs of disease and a greater mortality than younger pigs6. The suggestion that pigs could develop immunity to re- infection with L.intracellularis came from the observa- tion of two sequential outbreaks of PHE in a minimal dis- ease pig farm 7• The first outbreak affected highly susceptible breeding sows and boars of all ages, but was controlled with the addition of antibiotics to the feed. Seven weeks later the second outbreak occurred in gilts and young boars introduced into the breeding unit just before, during or after the in-feed medication was adminis- tered. It is probable that antibiotics in the feed of young pigs prevented infection and the development of immu- nity, and maintained susceptibility to L.intracellularis until these pigs were moved into the breeding unit and off feed containing medication. Strategically medicating pigs with antibiotics in combi- nation with exposure to L.intracellularis could permit the development of immunity to L.intracellularis while avoid- ing clinical signs of disease. One possible strategy would involve treating L.intracellularis-infected pigs with high levels of antibiotics in-feed once clinical signs are ob- served. A modification of this strategy successfully con- 2001 Allen D. Leman Swine Conference trolled PHE in newly introduced breeding animals. Pigs were exposed to infection for 21 days, then treated with antibiotics, before mixing the new pigs into the herd8. An alternative strategy to control PPE (ileitis) recom- mends continuous medication of diets with low levels of antibiotics. When the pigs are exposed to L.intracellularis, the antibiotics may subdue disease and allow subclinical infection and the development of immunity to re-infec- tion9• In this study we investigated whether pigs previously in- fected with L.intracellularis were immune to L.intracellularis on further exposure. The study also aimed to identify antibiotic medication strategies that allowed the development of immunity to L.intracellularis, but avoided clinical signs of disease. In order to develop strategic medication protocols to in- duce immunity to L.intracellularis in commercial pig farms, we also needed to investigate the effect of varying the dose of bacteria on immunity to L.intracellularis. It is probable that the ingestion of infectious feces by suscep- tible pigs would deliver lower doses of L. intracellularis compared with our experimental inoculations. The aim of the last experiment was to determine the initial mini- mum dose of L. intracellularis necessary to induce im- munity to a higher dose of L. intracellularis on re-inocu- lation. Demonstration of L.intracellularis infection in weaned pigs A reliable model for the experimental infection of pigs with L.intracellularis was developed to enable the inves- tigation of immunity to L.intracellularis. Previous experi- ments suggested that Large White, Landrace cross pigs were susceptible to L.intracellularis infection and devel- oped clinical signs of disease. Piglets were weaned at 3- 4 weeks of age into individual pens or weaner crates with solid walls and elevated floors to minimise the possible transmission of infection. L.intracellularis bacteria were extracted from homog- enized PHE-affected tissue as described previouslyI° and an aliquot of the bacterial suspension was stained by an indirect immunofluorescent assay to enumerate the num- 115 A. M. Collins ber· of L.intracellularis per milliliter of inoculum. Pigs were orally inoculated with 2SmL of bacterial suspen- sion containing between 109 and 10111 L.intracellularis via a tube passed well into the esophagus. Pigs were monitored for clinical signs of disease includ- ing diarrhea and reduced weekly weight gain. Blood samples were collected weekly for the detection of serum IgG antibodies against L.intracellularis, by an indirect immunofluorescence test (IFAT)". Fecal samples were collected twice weekly for peR amplification of L.intracellularis DNN2 and immunofluorescent staining of fecal smears 13. Feces and blood were collected prior to oral inoculation to verify that pigs were naIve to L.intracellularis. In the initial experiments one inoculated and one control pig was euthanased at 21 days post-in- oculation (pi), and sections of the ileum and colon were preserved in buffered formalin for hematoxylin and eosin staining. A temporal pattern of L.intracellularis infection was iden- tified using the above infection model. Pigs orally inocu- lated with between 109 and 1010 L.intracellularis devel- oped clinical signs of ileitis including diarrhea and reduced weight gains between 14 and 21 days pi. Prior to clinical signs becoming apparent, peR and immunofluorescent staining demonstrated consistent fecal shedding of L.intracellularis from 7 days pi onwards. Following the development of clinical signs, histologic lesions of PPE were observed in the terminal ileum of the inoculated pig necropsied at 21 days pi. The development of an immune response to L.intracellularis was evident between 3 and 8 weeks pi. Demonstration of immunity to L.intracellularis The development of immunity to re-infection with L.intracellularis was tested by re-inoculating previously infected pigs after the fecal shedding of L.intracellularis had ceased to be detected. Re-inoculated pigs were ob- served for clinical signs of disease, and the feces and se- rum were monitored for evidence of L.intracellularis in- fection. Pigs previously infected with L.intracellularis failed to shed detectable numbers of L.intracellularis in their feces and did not develop clinical signs of disease post-secondary inoculation. The absence of L.intracellularis in the feces of re-inoculated pigs sug- gests that L.intracellularis were inactivated before entry and colonization of mucosal cells. IgG producing plasma cells are directed to peripheral ar- eas, where their product can be detected by serology. The IgA-producing plasma cells are directed back to the LP, where secreted IgA is translocated through the epithelium and is available to bind to previously experienced anti- gens in the intestinal lumen. It is probable that secretory IgA specific for L.intracellularis was present in the intes- tinal lumen when the study pigs were re-inoculated. This secretory IgA would prevent the entry of L.intracellularis into mucosal cells and reduce the secondary immune re- sponse to L.intracellularis. Some pigs did develop a secondary serological response to L.intracellularis. These pigs may have been respond- ing to the L.intracellularis antigen in the intestinal lumen or may have become infected with a low level of L.intracellularis. If re-infection did occur, the number of L.intracellularis shed in the feces was lower than the de- tection sensitivity of the peR (approximately 4 x 102 L.intracellularislgram of feces). The development of immunity to L.intracellularis in pigs medicated with antibiotics Antibiotic medication was introduced into the develop- ment of immunity model described previously to reduce clinical signs of disease. Antibiotics were added either before or after the primary inoculation to modify the L.intracellularis infection. Diets containing oxytetracy- cline at SOppm, IOOppm, 300ppm, and 600ppm were sup- plied to groups of pigs four days prior to oral inoculation with L.intracellularis. Tylosin at SOppm and 100ppm, chlortetracycline at 400ppm, and olaquindox at 2Sppm, SOppm, and 100ppm were also supplied in-feed to groups of pigs 4 days prior to inoculation with L.intracellularis. Another group of pigs was inoculated with L.intracellularis before antibiotic medication with 400ppm of chlortetracycline from 14 to 24 days pi. Sepa- rate groups of pigs were maintained on non-medicated feed for all of these experiments to verify the infectivity of each inoculum. All of the pigs were orally inoculated with a sufficient number of L.intracellularis to cause clinical signs of ile- itis in unmedicated pigs. Pigs were monitored for clinical signs of disease, evidence of fecal shedding of L.intracellularis and a serological immune response against L.intracellularis as described in the original in- fection model. The initial immune response to L.intracellularis would Infection and the development of immunity were affected probably be generated in the mesenteric lymph nodes in three different ways in pigs continuously medicated (MLN), where bacteria that pass through the intestinal with antibiotics prior to inoculation with L.intracellularis. epithelium arrive via the lamina propria (LP). B cells in Some concentrations of antibiotics prevented the MLN are differentiated for IgA and IgG production in L.intracellularis infection and disease in all pigs and ex- response to antigenic stimulation. The small number of tended the susceptibility of pigs to L.intracellularis until J /6 2001 Allen D. Leman Swine Conference the antibiotic was removed. Pigs maintained on feed medi- cated with oxytetracycline at 300 and 600ppm, chlortet- racycline at 400ppm, or olaquindox at 100ppm were all totally protected from the first experimental dose of L.intracellulari.l', and did not develop immunity to re-in- fection with L.intracellulari.l'. On re-inoculation, all of these previously uninfected pigs became infected with L.intracellularis and developed clinical signs of disease, developing the same temporal pattern of infection as non- medicated pigs. Pigs maintained on non-medicated feed or pigs medicated with oxytetracycline at 50 or 100ppm, or 50ppm tylosin, were not protected and all became infected with L.intracellularis. These pigs shed L.intracellularis in their feces and developed a serological response against L.intracellularis at similar time points post-inoculation. Pigs medicated with relatively low levels of oxytetracy- cline or tylosin developed less severe clinical signs of disease than the pigs fed non-medicated diets. All of these previously infected pigs were immune to re-infection with L. intracellularis. The third situation involved groups of pigs that differed in their response to the primary inoculation and therefore also to the development of immunity. Diets containing olaquindox at 25 or 50ppm, or tylosin at 100ppm, did not prevent every pig from becoming infected with L.intracellularis. Fecal shedding of L. intracellularis and the development of an immune response was delayed for up to 2 weeks in those pigs that did become infected com- pared with non-medicated pigs. Pigs that had become in- fected with L.intracellularis following primary inocula- tion were immune to re-infection, regardless of the in-feed medication. Pigs on these medications that had been pro- tected by the above medications post-primary inocula- tion (i.e., had not shed L.intracellularis in their feces) were not immune to L.intracellularis, but were susceptible to L.intracellularis on secondary inoculation. The most successful strategies to induce the development of immunity to L. intracellularis allowed subclinical in- fection of pigs that were continuously medicated with low levels of antibiotics. The level of antibiotic medication necessary to allow subclinical infection and immunity may also depend on the level of exposure of pigs to L. intracellularis. Strategic antibiotic medication to induce immunity to L.intracellularis The treatment of pigs with 400ppm of chlortetracycline in-feed from 14 to 24 days pi terminated the fecal shed- ding of L.intracellularis and suppressed the primary se- rological immune response to L.intracellularis. Clinical signs of disease were also abbreviated and reduced in se- 200 I Allen D. Leman Swine Conference Immunity to Lawsonia intracellularis verity in pigs medicated with 400ppm chlortetracycline compared with non-medicated pigs. The development of immunity to re-infection with L.intracellularis was demonstrated following re-exposure, when fecal shedding of L.intracellularis was not detected. An anamnestic serological IgG immune response to L.intracellularis was detected in most of the pigs. The intensity of the secondary immune response was greatest in the pigs that had not developed a detectable IgG im- mune response post-primary inoculation. Implementing this medication strategy to control ileitis has many potential problems with regards to synchroniz- ing the timing of L.intracellularis infection and also the introduction of antibiotic medication. If medication is delayed beyond] 4 days after exposure, pigs may develop more severe clinical signs of disease. If medication is in- troduced before the initiation of an immune response to L.intracellularis, pigs may not develop immunity to re- infection. Variation in the dose of L.intracellularis may also affect the temporal pattern of L.intracelllllaris infection. The effect of dose on the development of immunity to L.intracellularis It was important to determine if natural exposures to L.intracelllllaris (via infectious feces) were sufficient to induce immunity to re-infection with L.intracellularis. It was previously calculated that experimentally infected pigs excrete between 5( 1 O~ and 7 (108 L.intracellularis per gram offecesl~. The aim of this experiment was to deter- mine the minimum dose of L. intracellularis necessary to induce immunity to a higher dose of L. intracellularis on re-inoculation. Five groups of pigs were inoculated with variable num- bers of L.intracellularis. Group I pigs were not inocu- lated. Group 2,3,4, and 5 pigs were inoculated with 2( 103, 2(105, 2(107, and 2(10 10 L. intracellularis, respectively. Pigs were observed daily for clinical signs of disease and infection was monitored by serology and fecal PCR for 10 weeks pi. At 70 days pi, all Group 2 to 5 pigs and a sixth group of susceptible recently weaned pigs were orally inoculated with a suspension of homogenized PHE-affected mucosa containing approximately I x 1010 L. intracellular!s. Clini- cal signs of disease and infection were monitored as de- scribed above. Severe clinical signs of ileitis (PPE) were observed in the pigs inoculated with 1010 L. intracellularis (Group 5). Persistent diarrhea between 14 and 26 days pi necessi- tated the treatment of a number of Group 5 pigs with an- tibiotics from 24 days pi. Substantial average weekly weight gain reductions were observed in Group 5 pigs between 2 and 4 weeks pi (Figure 1). Milder clinical signs 117 A. M. Collins Figure 1. The average weekly weight gain of pigs inoculated with 1010 (Group 5), 107 (Group 4), 105 (Group 3), 103 (Group 2) L.intrace//u/aris, or uninoculated pigs (Group 1). [--. groupl .. group3 x group4 7 - 5 ~-.----- -------- - . ---- --- --- -------- -- ~;~~~_:::=-~- 4 ______ ~.:;50n~~=-~~~