Studies on the transmissibility of pathogenic-organisms to liver by migrating larvae of liver fluke and hookworm
Introduction and Object
Wykoff and Lepes(1967) found that the excysted larvae of Clonorchis sinensis in the duodenum were capable of penetrating the intestinal wall and reaching the liver by the portal system. This indicates that the excysted larvae may reach the bile duct through the portal vein or peritoneal route.
It would be interesting to know whether the excysted or hatched larvae of parasites in the intestine could carry bacterial flora from the intestine to the liver during their migration, and thus causing hepatitis. It is an already known fact that the orally infected hookworm and ascaris larvae migrate into before lung circulation and cause temporary hepatic inflammation.
The present study has been designed to confirm whether the liver fluke or hookworm, which are the common parasites in Korea, could carry the pathogenic bacteria from intestine to liver.
Materials and Methods
Male rabbits weighing about 0.8∼2.0kg body weight were used as experimental animals. The metacercariae of Clonorchis sinensis were collected from raw fish by digestion method with artificial gastric juice, and 900∼1,200 of them were introduced directly into stomach by 5 mm diameter polyethylene tube. On the planned
date (1∼7th day), animals were sacrificed. The abdominal cavity was washed with saline solution. The solution was centrifuged in order to examine whether the sediments contained the adolescaria. The liver, small intestine and mesenterium and other organs in abdominal cavity were separated surgically and washed gently with
saline solution, and the solutions were also subjected to examination for the larvae. The liver tissues and the common bile ducts were examined for larvae.
In another experiment 15∼90 days old Clonorchis sinensis from the bile ducts were collected and introduced directly into the abdominal cavity of rabbits. After a designated date, the abdominal cavity and organs were carefully examined to fine the introduced worms. The contents of ducts were also examined. The area of the liver tissues where the worm was attached was sectioned for pathological and bacteriological examination.
In the 3rd experiment the young worms were collected from the bile ducts of rabbits which were infected with Clonorchis sinensis 11, 16 and 21 days before. The surface of the liver was incised and the collected worms were introduced into the wound, then covered gently with the outer lobe of the wounded liver and the abdominal wall was closed by routine surgical procedure. Three, six and ten days later, the animals were sacrificed, and the inoculated area was examined as above.
In the 4th experiment adult worms of Clonorchis sinensis were incubated in the saline containing Staphylococcus aureus for 30 hours at 25。C∼26。C. The surface of the rabbit liver was incised and the living adult worms which were incubated in the Staphylococcus aureus containing saline solution were introduced in the wound. Three days later, the inoculated area of liver was examined pathologically and bacteriologically. Intestinal contents of the Clonorchis sinensis from liver tissues were cultured in Nutrient-Agar plate and examined bacteriologically.
Cutaneous infection: Laboratory cultured rhabditoid larvae of cainne hookworm were incubated in normal tap water containing abundant Diplococcus Pneumoniae. Mice were narcotized with ether and fixed on the board. Abdominal surface of the mice was shaved following soap washing and 75% alcohol disinfection. 500∼1,000
filariform larvae suspension were smeared on the shaved area and left until they dried. On the 4∼8th day after the infection, the mice were sacrificed and the liver was examined patholofically and bacteriologically.
Oral infection: Rhabditoid larvae of canine hookworm were incubated in normal tap water containing Staphylococcus albus or Staphyococcu aureus. The larvae which developed to filariform stage in the media were fed to mice orally, and 1∼5 days later, the livers were examined pathologically and bacteriologically. Methylene blue and Gram's stain were applied.
A. Clonorchis sinensis
Recovery rats of larvae in the abdominal cavity of rabbits
One to seven days after the administration adolescariae were recovered from the abdominal cavity in less than 1% of the total number of metacercariae given. Generally, 1∼6 larvae were found from each animal which was given 900∼1,000 metacercariae, though many larvae were already found in the common bile ducts or remained still in intestine.
Fate of Clonorchis sinensis in abdominal cavity
The young or mature worms which were introduced directly into the abdominal cavity were examined 15, 32, 40 and 42 days after the inoculation. Several larvae were found on the surface of liver in four animals. All the worms on the surface of the liver were dead and the biopsied liver tissues on the area where the worms were attached showed no pathological change. Two of them were between bile duct and liver tissue but pus cell infiltration surrounding them was observed. In every case, pus cell infiltration was found in the peripheral portion of the liver and
pus nodules on the surface of intestine and mesentery. The nodule in the intestinal wall contained the eggs of Clonorchis sinesis. Two worms in the abdominal cavity were still alive. From the above results it is suggested that larvae of Clonorchis sinensis were capable of penetrating the intestinal wall and reaching the organs in th abdominal cavity and surviving for 15∼42 days, but they were unable to penetrate the organs. No bacterial flora appeared from the lesions by culture method.
Fate of Clonorchis sinensis which was inoculated into the peripheral region or liver
Small abscess was observed at the same area. Microscopically, the area became edematous and the vessel in the peripheral region were dilate. The parasites became necrotic and amorphous. Pathologically the lesions appeared as eosinophilic masses, and neutrophile leukocytes were infiltrated surrounding the masses. In some cases, the dead worms were found apart from the original place of inoculation but no leukocyte infiltration was found. There was linear infiltration between the original site and the portion where the dead worm was found. The distance from the capsule varied from 0 to 4 mm. Sometimes, the eggs of Clonorchis sinensis were also found. In all cases, there were no living worms in liver tissues and hepatic ducts.
In all cases, the bacteriological examination was negative.
Do Clonorchis sinensis transfer the micro-organism?
Five adult worms of Clonorchis sinensis were incubated in the saline solution containing Staphylococcus aureus. The intestinal contents of these worms were cultured in the Nutrient-Agar plate and examined by Methylene blue and Gram's stain. The area of liver tissue where the Clonorchis sinesis were inoculated showed
on inflammatory changes after the 3 days of inoculation but Staphylococcus aureus was found in the culture media with which the pieces of liver tissues were smeared.
Cutaneous infection: Four to eight days after the cutaneous infection of Ancylostoma caninum, the mice were sacrificed. Grossly, there was no abnormal finding in liver. The in four out of six examined. The micro-organism were confirmed as the same species of Diplococcus pneumoniae which were grown in the hookworm culture media.
Oral infection: 1,000 filari-form larvae of Ancylostoma caninum were given orally. 24 hours later, the mice were sacrificed and the pieces of liver tissue were smeared on the Nutrient-Agar plate. After 50 hours at 36。C, the bacterial colonies were examined bacteriologically. Staphylococcus albus was found from two out of four samples. Grossly there micro-abscesses which were infiltrated by leukocytes. The larvae were also found from other portions of liver tissues and they were surrounded by yellow colored material.
In another experiment, a combination of Ancylostoma duodenale and Staphylococcus aureus was fed to mice. The mice were sacrificed five days the oral administration of Ancylostoma duodenale cultivated in the madia containing Staphylococcus aureus.
The liver pieces were examined routinely. The larvae cultivated in normal tap water which contained no Staphylococcus aureus was used as control. In the experimental mouse, the cocci appeared in the liver. Pathologically, microabscesses infiltrated with neutrophile leukocytes were found, but there was no manifestation of inflammatory change due to the larvae penetration. Haemorrhage appeared only where the larvae were found.
In order to confirm whether the migrating larvae of parasites could carry pathogenic organisms into liver and cause hepatitis, a series of experiments has been carried out. The summary of the results is as follow:
1. Clonorchis sinensis
A few of the excysted larvae of Clonorchis sinensis penetrated into peritoneal cavity but they could not penetrate the liver tissues. The artificially introduced Clonorchis sinensis in the tissues were all destroyed within 3∼5days. The was no manifestation of diffuse inflammatory change due to the inoculation of the parasite, though the sampled micro-organisms, Staphylococcus aureus, were confirmed from the surrounding area.
The larvae carried pathogenic organisms to liver tissues either by cutaneous or oral infection, but there was no manifestation of hepatitis due to the micro-organisms; Diplococcus Pneumoniae, Staphylococcus albus et aureus.
In conclusion, it is indicated that liver fluke and hookworm may transmit pathogenic organisms to the liver during their migration.