Comparison of intestinal mucosa homogenate and pure culture of the homologous Lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine

Little information is available on reproduction of proliferative enteropathy (PE) using a virulent pure culture of Lawsonia intracellularis. Reproduction of the disease using PE-diseased mucosa homogenates, however, is well-characterized. The aims of this study were to evaluate and compare clinical signs, growth performance and the severity of lesions in pigs inoculated with intestinal mucosa homogenate or pure culture of the homologous L. intracellularis isolate. Five-week-old pigs were inoculated with pure culture of L. intracellularis (isolate PHE/MN1-00; n=10), PE-diseased mucosa (n=10), or control media (n=4). The L. intracellularis isolate PHE/MN1-00 used in the pure culture inoculum was extracted from a fragment of the same intestine used to prepare the mucosa homogenate. Clinical signs and growth performance were evaluated throughout the study. Fecal shedding was evaluated in all animals weekly during the experiment. All animals were euthanized 22 days post-inoculation, the intestines were examined grossly and histologically. Results showed that both the infection procedures reproduced clinical disease, macroscopic and histologic lesions typical of PE. Fecal shedding was detected in animals in both challenge groups. In conclusion, the L. intracellularis isolate PHE/MN1-00 reproduces typical clinical signs and lesions of PE similar to the homologous infection with an intestinal mucosa homogenate.     

Effect of dietary inclusion of distillers dried grains with solubles, soybean hulls, or a polyclonal antibody product on the ability of growing pigs to resist a Lawsonia intracellularis challenge

An experiment was conducted to determine if dietary inclusion of distillers dried grains with solubles (DDGS), soybean hulls, or soybean hulls sprayed with an egg-based, polyclonal antibody product would reduce the incidence or severity of infection, or both, in growing pigs after a Lawsonia intracellularis challenge. One hundred 17-d-old weaned pigs were blocked by sex, ancestry, and BW, and randomly allotted to 1 of 5 treatment groups: negative control, unchallenged, corn-soy diet; positive control, challenged, corn-soy diet; 20% DDGS diet (D), challenged; 5% soybean hulls diet (SH), challenged; and SH sprayed with a polyclonal antibody product diet, challenged. Challenged pigs were orally inoculated with 6.4 x 10(8) L. intracellularis organisms after a 4-wk prechallenge feeding period. On d 21 postchallenge, pigs were euthanized, lesions of intestinal mucosa were evaluated, and ileal tissue samples were analyzed by immunohistochemistry to determine the presence and proliferation rate of L. intracellularis. Challenging pigs with L. intracellularis reduced growth rate, feed intake, and efficiency of gain (P < 0.02) and increased the proportion of internal organ weights relative to BW (P < 0.01). Dietary treatment did not affect growth performance pre- or postchallenge (P > 0.10). Heart, empty stomach, and liver weights were similar among dietary treatments (P > 0.10). Weight of the large intestine as a percentage of BW was increased in D and SH pigs compared with positive control pigs (P < 0.05). Lesion length, prevalence, and severity, and fecal shedding of L. intracellularis were primarily unaffected by dietary treatment (P > 0.10), although ileal lesion length and severity observed tended to be greater in the SH sprayed with polyclonal antibody product diet vs. the D pigs (P < 0.10). Results from a previous study indicated that diet composition may affect length, severity, and prevalence of lesions caused by L. intracellularis in growing pigs subjected to a moderate challenge. However, beneficial results were not observed by feeding the dietary treatments used in this study.     

Colonisation and shedding of Lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms

Lawsonia intracellularis is an intracellular bacterium causing proliferative enteropathy in various animal species, and is considered an economically important pathogen of pigs. Rats and mice have been implicated as external vectors for a wide range of pig pathogens, including L. intracellularis. Previous studies have demonstrated L. intracellularis infection and proliferative enteropathy in rodents, but did not show the duration of shedding or the number of L. intracellularis shed by infected rodents, and therefore the infection risk that rodents pose to pigs. In this study, the number of L. intracellularis shed in the faeces and intestinal mucosa of wild rats trapped on pig farms was determined by a quantitative real time polymerase chain reaction assay. The prevalence of L. intracellularis in wild rats trapped on pig farms with endemic proliferative enteropathy (PE) was very high (≥ 70.6%), and large numbers of L. intracellularis were shed (10(10)/g of faeces) in a small proportion of wild rats. The duration of colonisation in laboratory rats and mice challenged with porcine isolates of L. intracellularis was also shown. Faecal shedding of L. intracellularis persisted for 14-21 days in rats and mice that were mildly affected with histological lesions of PE. The humoral immune response to L. intracellularis persisted for 40 days in both species. This study demonstrates that rodents may be an important reservoir of L. intracellularis on piggeries, and hence rodent control is important in disease eradication programs on pig farms.     

Comparison of different methods for diagnosis of porcine proliferative enteropathy

The objectives of this study were: (1) to compare 2 methods of serology; (2) to compare 3 histologic techniques; and (3) to compare 2 methods of detecting shedding in pigs experimentally challenged with Lawsonia intracellularis. The sensitivities of these tests were determined by the detection of infection. Forty 5-week-old pigs were inoculated on day 0 with intestinal homogenate from pigs with proliferative enteropathy (PE). Clinical evaluation was done on day 7 and daily from day 14 to 28 postinoculation. Fecal shedding of L. intracellularis was monitored by use of polymerase chain reaction (PCR) analysis and immunoperoxidase staining at 7-day intervals. Serum was obtained on days 0 and 28 for serologic testing by glass slide and tissue culture indirect fluorescent antibody tests. At euthanasia on day 28, gross intestinal lesions were evaluated and ileum samples collected for histologic analyses. Ileal histologic sections from each animal were stained by hematoxylin and eosin, Warthin-Starry silver stain, and immunohistochemistry (IHC). Of the 40 pigs, 36 had gross lesions typical of PE at necropsy. The percentage of agreement between the 2 serologic methods was 94.4%. Immunoperoxidase stain of fecal smears was more sensitive than PCR for detecting fecal shedding, especially on day 21 (89.5% and 60.5%, respectively) and day 28 (59.4% and 37.5%, respectively) post-inoculation. The IHC stain was much more sensitive for detecting infection than the routinely used hematoxylin and eosin and Warthin-Starry silver stains. In conclusion, in experimentally infected pigs, both serologic methods were appropriate techniques for detecting infection. For fecal samples, PCR has low sensitivity. Immunohistochemistry is the best diagnostic tool for formalin-fixed samples.     

Effect of including distillers dried grains with solubles in the diet, with or without antimicrobial regimen, on the ability of growing pigs to resist a Lawsonia intracellularis challenge

A disease challenge experiment was conducted to determine if including 10% dried distillers grains with solubles (DDGS) in the diet, with or without antimicrobial supplementation, reduces the incidence or severity, or both, of intestinal lesions in growing pigs after an Lawsonia intracellularis challenge. One hundred 17-d-old weaned pigs were blocked by sex, ancestry, and BW and randomly allotted to 1 of 5 treatment groups: negative control, unchallenged, corn-soy diet; positive control, challenged, corn-soy diet; 10% DDGS diet, challenged; positive control with antimicrobial regimen, challenged; and 10% DDGS diet with antimicrobial regimen, challenged. For antimicrobial-supplemented treatments, diets contained 33 ppm bacitracin methylene disalicylate throughout the experiment, with chlortetracycline (Aureomycin) pulsed at 550 ppm from d 3 prechallenge to d 11 postchallenge. Challenged pigs were orally inoculated with 8.0 x 10(8) L. intracellularis organisms after a 4-wk prechallenge period. On d 21 postchallenge, pigs were euthanized, lesions of intestinal mucosa were evaluated, and ileal tissue samples were analyzed by immunohistochemistry to determine the presence and proliferation rate of L. intracellularis. Compared with other dietary treatments, feeding a diet containing 10% DDGS reduced ileum and colon lesion length and prevalence (P < 0.05) and reduced severity of lesions in the ileum (P < 0.05) and colon (P < 0.10) in challenged pigs. Compared with other challenged pigs, those fed the diet containing the antimicrobial regimen had a lower prevalence and severity of lesions in the jejunum (P < 0.05) and tended to have reduced total tract lesion length (P = 0.11). Compared with other challenged pigs, pigs on the 10% DDGS diet with antimicrobial regimen exhibited no differences in length, severity, or prevalence of lesions (P > 0.15), but fecal shedding of L. intracellularis was reduced on d 14 postchallenge (P < 0.05). No dietary effects on fecal shedding were observed by d 20 postchallenge (P > 0.10). The proportion of cells infected with L. intracellularis was reduced when DDGS (P = 0.05) or antimicrobial (P = 0.10) diets were fed. Under the conditions of this experiment, dietary inclusion of 10% DDGS appears to provide some benefit to growing pigs subjected to a moderate L. intracellularis challenge, similar to those of a currently approved antimicrobial regimen.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.     

Primary infection protects pigs against re-infection with Lawsonia intracellularis in experimental challenge studies

In two separate trials pigs were experimentally infected with Lawsonia intracellularis at 5-6 weeks of age followed by antibiotic treatment and resolution of the primary infection and then re-inoculated at 12-13 weeks of age. A treatment-control group of pigs received the primary infection and antibiotic treatment only, and served as control for the antibiotic treatment of the primary infection. A challenge-control group of pigs received the second inoculation dose only at 12-13 weeks of age to control infectivity of the challenge-dose and susceptibility of pigs to L. intracellularis at this age. Pigs were monitored for shedding of L. intracellularis in faeces by PCR, and for the development of antibodies and responses of acute phase proteins in serum. The presence of L. intracellularis antigen in the intestinal mucosa was examined in post mortem samples by immunohistochemistry. In both trials primary infected pigs were protected from infection after challenge inoculation as evidenced by absence of faecal shedding of L. intracellularis, lack of changes in acute phase protein concentrations after challenge and with low levels of bacterial antigen in the intestinal mucosa of re-inoculated pigs comparable to that of the treatment-control pigs. In contrast, challenge-control pigs shed L. intracellularis in faeces, had L. intracellularis antigen extensively present within all layers of the intestinal mucosa and developed a significant acute phase protein response in serum after the experimental infection. The acute phase protein response to L. intracellularis infection was detected as an increased rise in the serum concentrations of C-reactive protein and haptoglobin from day-6 post infection, and increased serum concentrations of haptoglobin were generally seen 2-3 weeks after inoculation both at 5-6 and 12-13 weeks of age. In conclusion substantial protection against L. intracellularis infection was found in the re-inoculated pigs in contrast to the development of infection in age-matched control pigs. The acute phase protein responses reflected both the observed protection against L. intracellularis infection upon secondary challenge and that increased resistance to the infection develops with age.     

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection

A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.     

Experimental fecal transmission of human cryptosporidia to pigs, and attempted treatment with an ornithine decarboxylase inhibitor

Fecal material collected from an immunologically deficient man with persistent cryptosporidia infection was stored in potassium dichromate for two weeks and then fed (inoculated) to newborn pigs. The six inoculated newborn pigs shed the organism in their feces starting four to five days afer inoculation and continuing for as long as 22 days after inoculation. Pigs which were killed and necropsied while shedding had cryptosporidia infection of ileum, cecum, and colon. Infected pigs had atrophied ileal villi and flattened irregular cecal and colonic epithelium. Uninoculated littermate controls remained free to the infection and had histologically normal intestinal tracts at necropsy. Treatment of three of the six inoculated pigs with the ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine, orally for ten days had no apparent effect on the infection.     

Infection dynamics of Lawsonia intracellularis in pig herds

Little information is known about the natural course and within-herd prevalence of porcine proliferative enteropathy caused by Lawsonia intracellularis. The objective of the study was to investigate the within-herd dynamics of naturally acquired L. intracellularis infection in pigs from weaning to slaughter. The study was designed as a longitudinal survey where 100 pigs from five herds were randomly selected at weaning (approximately 4 weeks of age). Every second week until slaughter (10-12 times, i.e. 20-24 weeks) the pigs were weighed and faecal as well as blood samples were collected. Faecal shedding of L. intracellularis was assessed by real time-PCR and sero-conversion by an indirect immunofluorescence antibody test (IFAT). Clinical disease was not reported but infection was present in all herds and the PCR assay indicated infection in 75% of pigs examined. Most L. intracellularis infected pigs were shedding at 10-12 weeks of age (22-29 kg) and shed for 2-6 successive weeks. After 18 weeks of age all shedding had ceased and re-infection at PCR detectable level was not seen. Variable L. intracellularis associated impact on growth rate was observed. Immediately before bacterial shedding and during early infection the average growth rate declined whereas a compensatory impact was observed during later infection and after bacterial shedding had ceased. The performance of the IFAT resembled the bacteriological test almost perfectly. Sero-conversion was first detected at 12-14 weeks of age. Relative to the bacterial shedding, the onset of sero-conversion was a little delayed, in general, most pigs had sero-converted 2 weeks after the first shedding. Once sero-converted, 92% of the pigs remained sero-positive over the entire survey period.     

Detection of Lawsonia intracellularis in the tonsils of pigs with proliferative enteropathy

The presence of Lawsonia intracellularis, the obligate intracellular bacterium causing proliferative enteropathy (PE), in the tonsils of pigs as a locus for infection or extraintestinal occurrence of the bacterium was investigated by PCR and immunohistochemistry. Tonsillar occurrence of L. intracellularis could be part of the pathogenesis of PE and an important risk factor in the spread of the disease. L. intracellularis was detected by only PCR in the tonsils of 2/32 pigs without PE at necropsy but with a clinical history of diarrhoea and detection of the bacterium in faeces 1 to 3 weeks prior to necropsy but not in four pigs with moderate PE lesions. However, L. intracellularis was detected in the tonsils of 4/9 pigs with PE complicated with necroses and in 4/4 pigs with proliferative haemorrhagic enteropathy in which L. intracellularis antigen also was demonstrated in tonsillar macrophages and as intact bacteria in the lumen of the crypts. The results show that L. intracellularis is detectable in the tonsils of pigs and that the tonsillar presence of L. intracellularis appears to be correlated to the severity of the intestinal lesions possibly as a result of local retention and not as part of the pathogenesis of PE.     

Diagnosis of porcine proliferative enteropathy: detection of Lawsonia intracellularis by pathological examinations, polymerase chain reaction and cell culture inoculation

A total of 21 pigs aged 7-17 weeks with clinical symptoms suggestive for Porcine Proliferative Enteropathy were examined for Lawsonia intracellularis by analysing the following parameters: (i) intestinal gross and histological lesions, (ii) presence of comma-shaped bacteria in enterocytes by Warthin-Starry and a modified Ziehl-Neelsen stain, (iii) PCR amplification of L. intracellularis DNA from intestinal mucosa by using two oligonucleotide primer pairs targeting a 255-bp DNA fragment of the 16S rDNA-gene and a 319-bp DNA fragment of the L. intracellularis chromosome. Specificity of PCR reactions was confirmed by using DNA extracted from the L. intracellularis reference strain N343 (ATCC 55672) as well as by DNA sequence comparisons of PCR amplification products with data bank entries. Intestinal gross lesion indicative for PPE were observed in 20 pigs (95.2%). For all 21 pigs, the L. intracellularis aetiology was confirmed by histological as well as bacterioscopical examinations. Specific PCR amplification products were obtained from 20 pigs (95.2%). Taking PCR positivity as the definite criterion, L. intracellularis was diagnosed in 20 pigs from 11 herds in seven Swiss cantons (Argovia, Berne, Fribourg, Grisons, Lucerne, Schwyz, Thurgovia). To grow L. intracellularisin vitro, the cell culture method of Lawson et al. (J. Clin. Microbiol. 1993: 31, 1136-1142) was adopted. Inocula prepared from heavily infected fresh and frozen ileal mucosa of 15 pigs were cultured in rat enterocytic IEC-18 cells (ATCC CRL 1589). Six cell culture passages of 10 days each were completed. The reference strain N343 was examined for cultivability, accordingly. Except for occasional specific PCR amplifications from cell cultures up to the second passage, any indications for growth of L. intracellularis in IEC-18 cells were not found.     

Effect of dietary inclusion of distillers dried grains with solubles on the ability of growing pigs to resist a Lawsonia intracellularis challenge

An experiment was conducted to determine if including distillers dried grains with solubles (DDGS) in the diet of growing pigs reduces the incidence or severity of infection after a Lawsonia intracellularis challenge. Eighty 17-d-old weaned pigs were blocked by sex, ancestry, and BW and randomly allotted to 1 of 4 treatment groups: negative control (NC), unchallenged, corn-soy diet; positive control (PC), challenged, corn-soy diet; 10% DDGS diet (10D), challenged; and 20% DDGS diet (20D), challenged. Challenged pigs were orally inoculated with 1.5 x 10(9) L. intracellularis organisms after a 4-wk prechallenge feeding period. On d 21 postchallenge, pigs were euthanized, lesions of intestinal mucosa were evaluated, and ileal tissue samples were analyzed by immunohistochemistry to determine the presence and proliferation rate of L. intracellularis. Compared with unchallenged pigs, challenging pigs with L. intracellularis reduced growth rate, feed intake, and efficiency of gain (P < 0.01) and increased gauntness (P < 0.05) and diarrhea (P < 0.01). Diet did not affect growth performance postchallenge (P > 0.40). Feeding 10 or 20% DDGS diets did not reduce lesion length, prevalence, proliferation of L. intracellularis, or severity of lesions (P > 0.10). Thus, dietary inclusion of DDGS did not reduce the incidence or severity of lesions under the conditions of a severe L. intracellularis challenge used in this study.     

Onset and duration of fecal shedding,cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis

Little is known about the humoral and, especially, cell-mediated immune response in pigs exposed to Lawsonia intracellularis. The objectives of this study were to investigate the onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or a commercial live vaccine strain of L. intracellularis. Twenty-four 5-week-old pigs were exposed to 4.4x10(9) organisms of a pathogenic L. intracellularis isolate PHE/MN1-00 (10 pigs), a L. intracellularis live attenuated vaccine strain (10 pigs) or sham inoculum (4 pigs). Fecal, serum and whole blood samples were collected from all animals before exposure and weekly up to 13 weeks post inoculation and tested by PCR, immunoperoxidase monolayer assay serology and an interferon-gamma assay, respectively. One animal from each group was euthanized on day 22 post exposure to confirm infection. Humoral and cell-mediated immune responses were initially detected 2 weeks after exposure in pigs challenged with the pathogenic isolate, and 5 and 4 weeks, respectively, in pigs exposed to the modified-live vaccine group. Humoral and cell-mediated immune responses were still detected in some pigs from both L. intracellularis exposed groups 13 weeks after exposure. Fecal shedding was initially detected 1 week and lasted, intermittently, 12 weeks post exposure in pigs challenged with the pathogenic isolate, while fecal shedding was first detected 2 weeks and lasted, also intermittently, 9 weeks after exposure to the vaccine. In summary, both pathogenic isolate challenged and vaccine exposed pigs demonstrated long-term shedding of and immune responses to L. intracellularis.     

Immunohistochemistry and polymerase chain reaction for the detection of Lawsonia intracellularis in porcine intestinal tissues with proliferative enteropathy

Detection method of Lawsonia intracellularis was studied in formalin-fixed paraffin-embedded intestinal tissues from 5 naturally infected pigs by immunohistochemistry with a monoclonal antibody against outer membrane protein of L. intracellularis. Warthin-Starry silver stain revealed clusters of argyrophilic, slightly curved rod-shaped organisms in the apical cytoplasm of enterocytes. Immunohistochemical staining with a L. intracellularis-specific monoclonal antibody confirmed the presence of the organism in the apical cytoplasm of hyperplastic enterocytes. The presence of L. intracellularis in the ileum of pig with proliferative enteropathy was confirmed by polymerase chain reaction (PCR) further on the basis of amplification of 319 base pair products specific for porcine L. intracellularis chromosomal DNA. Immunohistochemistry and PCR may be a complementary method to confirm the diagnosis of L. intracellularis infection in pigs.     

The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge

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.     

Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when the pigs were 2-4 weeks old maternally derived IgG against L. intracellularis was demonstrated by immunofluorescence antibody test in nine pigs whereas the bacterium was detected by PCR in faeces from six pigs. The maternally derived antibodies did not prevent pigs from becoming infected as seven pigs later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracellularis exposed pigs with or without current proliferative enteropathy.     

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.     

Porcine neonatal coccidiosis: evaluation of monensin as preventive therapy

In this study, we evaluated the efficacy of monensin as a preventive therapy for porcine neonatal coccidiosis. Fifteen three-day-old piglets were given 50,000 sporulated oocysts of Isospora suis and eight of them received 15 mg/kg of monensin orally every other day. Seven piglets served as normal controls. Fecal samples were collected and checked for oocyst shedding. At 18 days of age, piglets were euthanized and necropsied.

The onset of clinical signs was delayed in the treated group, but all inoculated piglets displayed anorexia, soft stool, or diarrheic feces. Treated piglets shed large numbers of oocysts in their feces (up to 201,200 oocysts per gram of feces). All infected piglets had lesions of villous atrophy in the jejunum and most of them were in the late atrophic or villous regrowth stages.

The results of this study suggest that monensin does not prevent clinical signs, oocyst shedding, and intestinal lesions caused by I. suis in neonatal piglets.

     

Isolation of Lawsonia intracellularis in Korea and reproduction of proliferative enteropathy in pigs and hamsters

Lawsonia intracellularis (L. intracellularis) was isolated from a Korean pig suffering acute proliferative enteropathy. In vitro culture conditions of L. intracellularis were established in McCoy cells. Pigs and hamsters experimentally infected with the pure culture of L. intracellularis reproduced clinical signs and intestinal lesions of proliferative enteropathy. The presence of L. intracellularis in the intestinal lesions was confirmed by immunohistochemistry with L. intracellularis-specific monoclonal antibodies.