Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Experimental infection with Mycobacterium avium, serotype 2, 2. Oral infection with large doses of M. avium

Twelve pigs were inoculated orally with Mycobacterium avium. The doses used were 0.5, 2 or 10 mg daily for 5 days, or 10, 50 or 180 mg once (1 mg = 37 × 10⁶ viable units). Two pigs were used per dose, 1 of which was sacrificed 3 days, the other 28/31 days after the last inoculation (Table 1).Three days after inoculation, M. avium was found in the tonsils and in the intestinal mucosa of all 6 pigs, and in the mesenteric lymph nodes of 4. Viable unit counts for tonsils and intestinal mucosa were highest in pigs inoculated with 180 mg×1 and 10 mg×5. Histopathologically these pigs showed activation of the lymphoid tissue in the tonsils, Peyer patches and mesenteric lymph nodes. Twenty-eight/31 days after inoculation a spreading of the infection had taken place in all pigs, most often to the liver, less frequently to the spleen and the lungs. The kidneys and the musculature were not infected (Table 4). A correlation was apparent between the size of dose and the number of viable organisms in the tissues. Divided doses gave about 10 times higher viable counts than a single dose with the same total number of organisms (Table 5).No gross lesions were found 28/31 days after inoculation. Microscopic granulomatous lesions were found in the tonsils of 6 pigs, in the intestinal mucosa of 4 pigs, in the mandibular and mesenteric lymph nodes of 6 pigs, in the retropharyngeal lymph nodes of 3 pigs, and less frequently in the parotid and hepatic lymph nodes (Table 3).Five of 6 pigs were weakly sensitive to avian tuberculin PPD, 1000 t.u. per dose, when tested 22/25 days after inoculation; 1 of these pigs cross-reacted to human tuberculin (Table 2).     

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.     

Experimental reproduction of porcine proliferative enteritis

Conventional crossbred pigs from different sources and of different weights were examined for susceptibility to porcine proliferative enteritis. The ileal mucosa of pigs with the disease was emulsified and suspended in Mueller-Hinton broth. Pigs weighing 15, 120 and 200 lb (6.8, 54.5 and 91 kg) (four pigs per group) were stressed and inoculated orally with 80 ml of emulsified proliferative ilea. Severe lesions of porcine proliferative enteritis were detected in three of the four pigs weighing 6.8 kg. Mild lesions were detected in two of the four pigs in each of the other two groups. Gross lesions consisted of reticulation of the serosa, and hyperaemia and thickening of the mucosa with either fibrin or blood clots adherent to the mucosal surface. Inflammation, numerous mitotic figures and epithelial cell proliferation were observed microscopically in the crypts. Silver stained sections revealed numerous comma-shaped organisms in the crypts of infected epithelial cells. Using this method, serial reproduction of the disease was accomplished through the passage of fresh and previously frozen inocula. The virulence of the freshly prepared inoculum increased with passage through the host, whereas the inoculum prepared from tissue that had previously been frozen showed a decrease in infectivity and virulence. These data provide strong evidence for the infectious nature of this disease.     

Immune responses after local administration of IgY loaded-PLGA microspheres in gut-associated lymphoid tissue in pigs

Oral vaccination of large animals using PLGA MS (poly(D,L-lactide-co-glycolide)microspheres) appeared to be more challenging than immunization of mice. The purpose of this study was to deliver to GALT an immunogenic model protein (IgY), free or encapsulated by spray-drying in PLGA MS, and to evaluate systemic immune response in SPF Large White pigs. Pigs were surgically processed for local administration of IgY in three sets of experiments. In two sets of experiments, administration was locally performed in temporary ligatured intestinal segments, in jejunal Peyer's patches and in mesenteric lymph nodes. In the third experiment, pigs received IgY via an intestinal cannula. Total IgY-specific antibodies were detected in the sera of pigs after a single local immunization, but not in the sera of cannulated pigs. The study of IgG1 and IgG2 isotypes indicated that PLGA MS are able to elicit a combined serum IgG2/G1 response with a predominance of IgG1 response when locally administered. PLGA MS can be a potential oral delivery system for antigen but our results underlined the difficulty to immunize large animals like pigs. Transposition of data between small and large animals appears to be complex and suggests that physiological features need to be considered to increase intestinal availability of oral encapsulated vaccines.     

A NEW TYPE OF RE-ENTRANT CANNULA DESIGNED FOR USE IN THE SMALL INTESTINE OF THE PIG

A new type of intestinal re-entrant cannula for the small intestine of pigs is described. It consisted of an intestinal tube joined to a stem bearing a flange and two perspex plugs ("maintenance plug" and "collection plug"). The cannula was inserted in the intestine through the incision on the antimesenteric side of the intestine and no transection of the intestine was required. No outside connection was present and the cannula extended only 1 to 2cm beyond the body. Cannulae were inserted in the duodenum and ileum of 5 pigs and these functioned satisfactorily for up to 3 months.     

Experimental Atrophic Rhinitis in Gnotobiotic Pigs

Twenty-nine caesarian derived colostrum deprived germfree pigs were reared in isolators in groups of three to four per isolator. At seven days of age each group was inoculated intranasally with one of four strains of Bordetella bronchiseptica (designated B, J, L and 55B), or Pseudomonas aeruginosa or a mucoid strain of Escherichia coli, all previously isolated from nasal mucus of pigs affected with clinical atrophic rhinitis. Another group was inoculated simultaneously with B. bronchiseptica B and Pasteurella multocida. The animals were observed for clinical signs of atrophic rhinitis and monitored bacteriologically at weekly intervals for seven weeks. Then they were bled for serology and killed and their respiratory organs examined for gross and histopathological lesions.

All of the pigs inoculated with the Bordetellae had inflammation of the nasal mucosa and developed positive serum antibody titers against all four of the Bordetella strains used in this study. Strain J caused sneezing and turbinate atrophy in three of four pigs. One of the three pigs inoculated with strain L died in ten days from bronchopneumonia and pericarditis and had turbinate atrophy. Strains B and B55 caused no turbinate atrophy, but two out of three pigs inoculated with both B. bronchiseptica B and P. multocida had turbinate atrophy. No nasal lesions were observed in the pigs inoculated with E. coli or P. aeruginosa or in the noninoculated germfree controls.

The results indicate a variation in the ability of different strains of B. bronchiseptica to cause turbinate atrophy in pigs and demonstrate that nasal infections by these organisms stimulate serum antibody response. Presence of P. multocida appears to increase the severity of the lesions. As the E. coli and Pseudomonas failed to produce atrophic rhinitis, they are probably of no significance as primary etiological agents in the atrophic rhinitis syndrome in swine.

     

Single and mixed infections of neonatal pigs with rotaviruses and enteroviruses: clinical signs and microscopic lesions.

Neonatal colostrum-deprived pigs were inoculated with cell-culture preparations of three rotaviruses and three enteroviruses, singly or in combination. The three enteroviruses established intestinal and systemic infection but did not induce diarrhea or intestinal lesions. The three rotaviruses produced severe enteric disease characterized by profuse watery diarrhea, dehydration and death. Villi were severely stunted. All three isolates were equally virulent. Inoculation with three different rotavirus-enterovirus combinations resulted in disease less severe than that produced by the rotaviruses alone. Intestinal lesions were less extensive and fewer pigs became moribund or died.     

Pharmacokinetics and tissue distribution of amoxicillin in healthy and Salmonella Typhimurium-inoculated pigs

OBJECTIVE: To determine pharmacokinetics and tissue distribution of amoxicillin in healthy and Salmonella Typhimurium-inoculated pigs. ANIMALS: 12 healthy pigs and 12 S Typhimurium-inoculated pigs. PROCEDURE: Concentration of amoxicillin in tissue was measured by use of high-performance liquid chromatography 4, 8, 12, and 24 hours after IM administration. Pharmacokinetic values of amoxicillin in plasma were assessed by use of a 1-compartment model with first-order absorption. RESULTS: Inoculation caused diarrhea and increased rectal temperature and WBC count. Absorption half-life was shorter in inoculated pigs (0.26 hours) than in healthy pigs (0.71 hours), and inoculated pigs had longer elimination half-life. Distribution ratios in healthy pigs ranged from 0.31 to 0.56 and in inoculated pigs ranged from 0.14 to 0.48. Ratios for distribution to intestinal mucosa ranged from 0.34 to 1.16 in healthy pigs and from 0.22 to 0.36 in inoculated pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Salmonella Typhimurium inoculation altered absorption of amoxicillin from the injection site and prolonged elimination half-life. However, distribution of amoxicillin to intestinal tract tissue was only affected to a minor degree.     

Relationship of porcine cytomegalovirus and B bronchiseptica to atrophic rhinitis in gnotobiotic piglets.

Both porcine cytomegalovirus (PCMV) and Bordetella bronchiseptica produced rhinitis and pneumonia when inoculated intranasally into young gnotobiotic pigs. With PCMV the nasal lesions were confirmed to the lamina propria, while Bordetella produced atrophy of the turbinate bones and hyperplasia and degeneration of the nasal epithelium. Some exacerbation of the lesions was observed in the nasal mucosa of pigs given both agents, but the degree of bone atrophy was not increased.     

Enterovirulence of enterotoxigenic Bacteroides fragilis in gnotobiotic pigs.

A porcine isolate of enterotoxigenic Bacteroides fragilis colonized the intestinal tract and caused watery, nonhemorrhagic diarrhea when given orally to 12, 1- to 2-day-old gnotobiotic pigs. Diarrhea occurred 2 to 3 days post-inoculation and continued throughout the 4 to 6 day post-inoculation period. Diarrheic pigs became mildly anorexic and dehydrated. They developed intestinal lesions characterized by swelling, vacuolation, and exfoliation of enterocytes, and crypt hyperplasia throughout the large intestine and, to a lesser extent, in the distal small intestine. Bacterial adherence to, or invasion of, the intestinal mucosa was not detected. A porcine isolate of nonenterotoxigenic B. fragilis was administered orally to six control pigs. The isolate colonized the intestinal tract, but the pigs did not develop clinical disease or intestinal lesions. The pathogenetic mechanism of the disease may involve mediation by a soluble enterotoxin (or toxins) elaborated by B. fragilis.     

Effects of experimenters and different blood sampling procedures on blood metabolite values in growing pigs.

The aims of the present study were firstly to verify if sera obtained by jugular venipuncture and those obtained from an anterior vena cava cannula were comparable and secondly to observe the effect of different experimenters on the biochemical profile of pigs. In the first experiment, 16 Yorkshire pigs with a mean weight of 48.9 +/- 2.3 kg were venipunctured using either a vacuum tube system or a syringe. The experiment was conducted on two separate days, seven days apart, in a crossover design experiment. At the time of obtaining blood via venipuncture, blood was also withdrawn from the jugular cannula. The tip of the latter was in the anterior vena cava. No effect of the sampling system was observed. However, a significant decrease in serum concentrations of Na, P and CO2 and a significant increase in anion gap, total protein, albumin, globulin, total bilirubin, aspartate amniotransferase, L-gamma glutamyl-transferase and creatine kinase were observed from blood samples obtained by venipuncture. In the second experiment, ten pigs from the first experiment were sampled by an inexperienced manipulator. Variations of the same magnitude as observed in the first experiment were observed in sera obtained by venipuncture and anterior vena cava but 18 of the 22 biochemical variables of sera obtained from the anterior vena cava were significantly different than those from the first experiment. The results show that blood samples obtained by jugular venipuncture in pigs can differ from samples obtained from an anterior vena cava cannula, specially in enzyme concentrations, and the dexterity of the experimenter may have a major effect on blood values.     

Tyzzer's disease in a dog.

A 5-week-old mixed-breed dog was examined because of emaciation and depression associated with chronic anorexia, diarrhea, and vomiting. Its rectal temperature was subnormal and it died on the day of admission. At necropsy, small focal lesions were distributed through the liver. Enteric alterations included catarrhal enteritis with fluid contents, excess production of mucus, and mucosal hyperemia. Microscopically, the hepatic lesions were disseminated foci of coagulative necrosis, with little or no associated inflammatory cell response. Numerous organisms morphologically consistent with Bacillus piliformis were demonstrated within viable hepatocytes at the periphery of the necrotic foci and in the intestinal mucosa. Numerous coccidial forms were found within the epithelial cells of the intestinal mucosa, which was focally necrotic.     

Technical note: a 2-stage cecal cannulation technique in standing horses

Cecal cannulation is necessary for sampling of intestinal contents for a variety of nutritional or digestive physiology studies. This report describes a 2-stage technique for permanent cecal cannulation in standing horses. For the first procedure, a right flank laparotomy is performed and a small pouch of the cecal base exteriorized and sutured to the body wall. The second procedure is performed approximately 1 wk later. During the second procedure, the exposed cecal pouch is removed and the cannula inserted. Ten horses were cannulated using this technique. After the first procedure, 1 horse developed a cecal impaction unresponsive to medical therapy and ruptured its cecum, whereas 2 other horses developed mild transient colic that responded to medical management. Insertion of the cecal cannula after creation of the stoma in the second procedure resulted in transient colic in 4 of 9 horses, but they responded to analgesic therapy in less than 24 h in all instances. The time to complete healing of the cannula site was approximately 30 d. The technique described in this report decreases the risk of peritonitis due to intestinal leakage and is technically easier to perform than previously described techniques.     

Effects of short-chain fatty acids and lactic acids on survival of Oesophagostomum dentatum in pigs

The direct influence of intracaecal infusion of short-chain fatty acids (SCFA) and lactic acids (LA) on already established Oesophagostomum dentatum infection in cannulated pigs was investigated. We tested the hypothesis that the previously discovered anti-parasitic effect of inulin is mediated through its metabolic products SCFA and LA by infusing into cannulated pigs these compounds in amounts approximating to those produced in the pigs large intestine and caecum during the metabolism of inulin. The experiment comprised of 18 pigs--2 groups of 9 pigs in each. The normal diet used in the experiment was based on barley flour with insoluble fibre from oat husk with added soybean meal, vitamins and minerals. After 2 weeks of adaptation to the diet all the pigs were inoculated with 6,000 infective larvae of O. dentatum. Six weeks later, surgery on all pigs was performed to install cannulas into caeci. At 7 weeks post-infection (p.i.) the SCFA and LA infusion was initiated in Group 1 (experimental) pigs; at the same time pigs in Group 2 (controls) were infused with saline. At week 10 p.i., all pigs were killed and their worm burdens determined. SCFA and LA infused pigs exhibited markedly reduced fecal egg counts and worm recoveries (98 and 92% reduction, respectively, compared to saline controls). The results from this study demonstrate that SCFA and LA have a significant negative influence on established O. dentatum infection in growing pigs. The results also show that the type of dietary carbohydrates fed and its intestinal degradation can yield metabolic by products that profoundly influence helminth survival.     

A Lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model

Transmission of Lawsonia intracellularis from experimentally inoculated pigs to naive swine was demonstrated in this study. The study was conducted using conventional pigs divided into three groups as follows: principles inoculated with L. intracellularis, sentinels, and controls. The pigs were inoculated and paired on 13 and 9 days post-inoculation with a sentinel pig for 7 days. Fecal samples and serum samples were collected throughout the study for polymerase chain reaction (PCR) and antibody testing by indirect fluorescent antibody techniques. After co-mingling, the inoculated group was necropsied; sentinel and control pigs were necropsied 7-14 days later. The intestinal tracts were evaluated grossly and microscopically for lesions. PCR was performed on intestinal mucosal scrapings and feces. Warthin-Starry and fluorescent antibody staining procedures were conducted to confirm colonization with L. intracellularis. Gross and microscopic lesions typical of porcine proliferative enteropathy (PPE) were observed in both the inoculated and sentinel groups. Transmission was demonstrated from inoculated principle pigs to sentinel pigs. PCR results detected cyclical shedding of L. intracellularis in the feces. Seroconversion occurred in pigs that were exposed to L. intracellularis. From this study, it was demonstrated that transmission of L. intracellularis can occur easily in an environment with experimentally infected pigs and that PCR can be a useful tool to monitor fecal shedding of the organism.     

Diagnosis and surgical management of vascular ectasia in a dog

CASE DESCRIPTION: An 8-year-old male Golden Retriever was evaluated because of an 8-week history of intermittent diarrhea with melena and hematochezia that were not responsive to medical treatment and resulted in severe anemia. CLINICAL FINDINGS: Exploratory celiotomy with intestinal and colonic biopsy revealed mild enterocolitis but did not result in diagnosis of the cause of melena and hematochezia. Endoscopy of the upper portion of the gastrointestinal tract and colonoscopy were performed. Multifocal areas of coalescing, tortuous mucosal blood vessels were observed in the cecum and all regions of the colon. A diagnosis of vascular ectasia (VE) was made on the basis of the endoscopic and histologic appearance of the lesions. TREATMENT AND OUTCOME: An ileorectal anastamosis was performed. Melena and hematochezia resolved within 3 days after surgery, and the anemia resolved within 6 weeks after surgery. Surgical resection of the cecum and colon and feeding of a highly digestible diet resulted in long-term (22 months) resolution of clinical signs. CLINICAL RELEVANCE: Initial exploratory celiotomy with intestinal and colonic biopsy failed to reveal the VE lesions responsible for the melena, hematochezia, and anemia. Endoscopic evaluation was necessary for detection of the colonic VE lesions. Surgical resection of the cecum and colon and feeding of a highly digestible diet may result in a favorable outcome in affected dogs.     

Studies on the pathogenesis of Salmonella typhimurium and Salmonella choleraesuis var kunzendorf infection in weanling pigs

Twenty-six 4-week-old pigs were randomly allotted to 4 groups: group 1--orally inoculated with Salmonella typhimurium; group 2--orally dosed with S choleraesuis; and groups 3 and 4, with surgically constructed intestinal loops--loops inoculated with either S typhimurium or S choleraesuis. One pig each from groups 1 and 2 was killed at 8, 12, 24, 48, 72, 96, and 120 hours after inoculation. One pig each from groups 3 and 4 was killed at 2, 4, 6, 8, 12, and 24 hours after intestinal loop inoculation. Inoculation of S typhimurium resulted in acute enterocolitis of variable severity, whereas inoculation of S choleraesuis resulted initially in septicemia followed by formation of large necrotic and ulcerative lesions in the colonic mucosa. The most consistent systemic lesion of S choleraesuis infection was interstitial pneumonia and multifocal hepatic necrosis. Salmonella typhimurium and S choleraesuis were ultrastructurally within enterocytes of ligated ileal loops. Intracellular bacteria were morphologically intact, occurred free in the cytoplasm and membrane bound, and caused no detectable cytotoxic effect to the cell. Both S typhimurium and S choleraesuis penetrated the intestinal mucosa and were isolated from mesenteric lymph nodes at 2 hours after inoculation.     

Panfibrinonecrotic colitis in a dog treated by subtotal colectomy

A dog with signs of small intestinal disease developed signs of severe large intestinal disease after being treated with multiple agents. Endoscopy and biopsy revealed total destruction of the colonic mucosa, which necessitated total parenteral nutrition plus subtotal colectomy. It was hypothesized that the multiple prior treatments led to an overgrowth of toxigenic or cytotoxic bacteria, which caused this colonic mucosal destruction.     

Intestinal crypt lesions associated with protein-losing enteropathy in the dog

Six dogs were diagnosed with protein losing enteropathy (PLE). There was no evidence of inappropriate inflammatory infiltrates or lymphangiectasia in multiple mucosal biopsies of the small intestine of 4 of the dogs. The 5th and 6th dogs had obvious lymphangiectasia and a moderate infiltrate of inflammatory cells in the intestinal mucosa. All 6 dogs had a large number of dilated intestinal crypts that were filled with mucus, sloughed epithelial cells, and/or inflammatory cells. Whether PLE occurs in these dogs because of protein lost from the dilated crypts into the intestinal lumen or whether the dilated crypts are a mucosal reaction due to another undetermined lesion that is responsible for alimentary tract protein loss is unknown. However, when large numbers of dilated intestinal crypts are present, they appear to be associated with PLE even if there are no other remarkable lesions in the intestinal mucosa.