Epidemiological studies on the pig enteritis　due to Lawsonia sp. and Clostridium spp. In Niigata Prefecture

This study was conducted an occurrence status investigation, pathological tests, bacteriological tests, and other tests for epidemiological research on an orally-infected digestive disease in pigs in Niigata Prefecture, i.e. pig enteritis due to Lawsonia sp. and Clostridium spp. that could cause serious economic loss. Lawsonia genus contains only one species Lawsonia intracellularis (Li), an obligate intracellular eubacterium that does not culture on artificial media. It is a relatively new species that was discovered in 1995 as the offending bacterium in proliferative enteritis. 1982 and 1984, an investigation was conducted on breeding pigs and the fattening pigs that died or were sent to the slaughterhouse after excreting dark reddish blood feces, and adenomatous hyperplasia of crypt epithelial cells were detected in the ileum mucosa of every pig. Curved bacilli, which dye with Warthin-Starry (WS) stain, were also found in hyperplastic crypt epithelial cell cytoplasm. The occurrence of acute proliferative hemorrhagic enteropathy (PHE) was thus confirmed in Niigata Prefecture. A hearing investigation conducted at 24 farms that breed the same kind of pigs in the Prefecture revealed a total of 40 pigs with PHE-like diseases in 12 farms during the period from the first occurrence of the disease in a breeding pig in June 1982 to September 1984. The occurrence was limited to the same kind of pigs and the incidence rate was 0.22%. Since then, acute PHE has been detected sporadically. Seventeen years after the first occurrence, an inspection of two-month old stunted piglets pathologically revealed adenomatous hyperplasia of crypt epithelial cells in the mucosa in the lower small bowel of each piglet, although there was individual variability in the ileum mucosa thickening. The WS staining highlighted the curved bacilli in crypt epithelial cell cytoplasm, and PCR test revealed the Li gene, indicating the first confirmation of chronic porcine proliferative enteritis (PPE) in Niigata Prefecture. Sixty-four piglets with chronic PPE were found in 2 farms in the following year of 2000, 76 piglets in 3 farms in 2001, and 129 piglets in 6 farms in 2002, demonstrating an upward trend of the disease. Spreading to the large intestine was observed at a high percentage, i.e. in 21 out of 24 cases. In an examination of the chronic diseases, it was found that PPE easily causes secondary infection toward diarrhea, poor development, and other symptoms, and has the potential to become a critica base disease leading to gastrointestinal infectious disease. The progress of Li gene analysis technology has advanced examination of disease sources. The Li gene was detected by the PCR test in the ileum mucosa with proliferative enteritis of 2-month old poorly developed piglets. The detection in addition to a histopathological test resulted in the diagnosis of chronic PPE, which indicated that PCR could be used for disease examination. The Li gene was detected in the feces of the examined pigs that experienced diarrhea, which suggested Nested PCR could be used for disease examination before death. An antibody test using the indirect fluorescence antibody (IFA) method gave positive results in 29 (90.6%) out of 32 farms and in 74 (77.1%) out of 96 pigs as of 2003, which indicated vast spreading of the antibody. With regard to the infection condition inside the farms, we found infection-positive pigs in 5 out of 6 breeding pigs. The fattening pigs became positive at the age of 4 months and over in all the farms, which indicates they were infected in infancy. The infection pattern was a chronic frequent pattern 1 characterized by a high positive rate of the breeding pigs. A retrospective study of conserved serum exhibited negative results until 1985 but positive results at 4 farms (44.4%) in 1989 and 2 (22.2%) in 1993, showing a moderate change in the infection status.However, the infection rate increased in 1998 with 8 farms (88.8%) being positive, and the infection was confirmed to have spread to many farms in Niigata Prefecture. Clostridium spp. infectious disease ware also investigated. These were due to Clostridium perfringens Type A (CPA), Clostridium perfringens Type C (CPC), and Clostridium novyi (Cn), which had a high death rate as Clostridium spp. causes digestive disease in pigs. The disease cases of CPA in the fattening farms showed a high death rate and 31 out of 32 hogs died after having fever and bloody diarrhea as major features. An autopsy revealed a black-water content in their appendices and colons, and the formation of a blackish false membrane as well as a number of immature trichuris. A biopsy revealed catarrhal fibrinous enteritis in the intestine and diphtheric enteritis with Trichuris suis parasitism in the large intestine. In a bacteriological study, a large amount of CPA that produces a toxin and Enterotoxin was found in the small and large intestines, which suggested that the diseases were porcine trichuriasis accompanied by CPA infection. The death rate of porcine trichuriasis is normally 0.48-29.4%, while both toxins caused the high rate in these cases. As an example of the high death rate of newborn piglets due to CPC, 32 out of 53 2-day old piglets born from 6 mother pigs died suddenly in a consistently managed farm that breeding 220 pigs. An examination revealed dark red jejunum, and CPC and its toxin in the aforementioned organs, and the piglets were diagnosed with necrotic enteritis caused by CPC. In an epidemiological study, CPC was isolated from 1 out of 2 formula feed samples and 1 out of 15 feces samples. An indirect hemagglutination antibody test was conducted using the isolated CPC as an antigen, and 16 (80%) out of 20 pigs showed an antibody level more than 4 times higher than the others when they were introduced and 13 (86.3%) out of 15 pigs showed a high antibody level when they had the disease, indicating that the antibody level was already high in most pigs when they were introduced as rearing pigs. In particular, 5 mother pigs in the farm where the diseased pigs were found showed a high level of GM36.8. A mass death of the breeding pigs was seen for example in the following case: A disease with the symptom of nasal bleeding developed in a group of 30 breeding pigs and 11 out of them died prematurely. Three dead pigs were examined. Their various organs had liquefied, and dense sponge-like air bubbles were observed under the liver capsule. A histological examination revealed notable necrotic change in every organ with a number of large bacillus, and Cn was found in liver smears using the fluorescence antibody technique. The Cn was separated from the organs of one of the three dead pigs using the gas jet method, and a VFA production performance test detected butyric acid and propionic acid, indicating it was Type A toxic strain. Nineteen living pigs had an indirect hemagglutination antibody value 128-512 times higher than the separated Cn-A. The mass death was terminated by muscle injection of ampicillin and antimicrobial supplementation to feed. Since the disease did not develop in young pigs and rearing pigs that were fed with antibiotic-supplemented feed, it was presumed that the cause of the disease was leftover feed. As described above, an epidemiological study was conducted on an orally-infected digestive disease of pigs caused by Lawsonia sp. and Clostridium spp. in Niigata Prefecture, as well as on the occurrence status and retention condition of the antibody. In Lawsonia bacterium disease, an invading tendency from acute to chronic type was found, the persistent existence of the Clostridium bacterium disease was confirmed and the origin of the infection source was identified