Experimental studies on tropism and resistance of the infective larvae of Trichostrongylus orientalis
Since Artgur Looss(1905) established the Genus: Trichostrongylus, about36 species of the genus have been described by several workers (Otsur, 1962). The natural hosts of Trichostrongylus are the herbirotous animals, but some of them have been reported infest to human body; T. orientalis, T. colubivorous animals, T. vitrinus, T. probolurus, T. axei and T. brevis(Otsuru, 1962).
Otsuru (1962) obtained 52, 461 worms from 24 autopsied cases and 50 infected cases. He analyzed them after morphological examination as follows: T. orientalis 25,444, T. axei 6, T.colubriformis 5, and concluded that the main species for human
infection in Japan was the T.orientalis. In Korea, T. orientalis has been known as the only species which infest to human body, and the prevalence seems rather high; 64.0∼72.8%(Mine 1914), 23.3∼35.3%(Kobayashi 1929), 22.6%(Soh, et al. 1961).
Due to the limited distribution of T. orientalis in Korea, Japan, China and some other neighboring countries, the biological studies have been achieved mainly from this area. According to the literature, Hasegawa(1993) was one of the initiator in this field. He tried to distinguish the biological characters with hookworm, which has similar life cycle with Trichostrongylidae but is belonged to Ancylostomatidae. However, there are many spaces to be conformed concerning the bionomics.
The present study has been desined to find and regulate the biological characteristics of the larva biology in outside and inside the human body.
Materlals and Methods
Eggs of Trichostrongylus orientalis were obtained from the feces of the infected cases by concentration method. The eggs were smeared on filter paper and applied the Harada's culture method. After 10∼14 days cultivation 30。C incubator, the 3rd
stage larvae were used for experiments.
Thermotropism: The infective larvae were pasted on a rectangular filter paper(Fig. 1). Oen end of the paper was put into water in concial tube which was conditioned 10。C, 30。C and 40。C. As control the larvae of Ancylostoma caninum were used. The number of the larvae which migrated down to the water was counted at the 1 hour interval for 6 hours.
For another experiment, two conical tubes were connected by a rectangular filterpaper making a 0.5cm bridge between the open mouth of the two tubes. Each side of the paper was dipped in the water of the tube and the larvae suspension was put on the bridge(Fig. 2). The tubes were kept separatly in the water bath of 10。C and 35。C.
Haemotropism: Agar-agar medium and 5, 20, 30 and 50% of human blood afar media were prepared. The agar-agar medium was put in the petri-dish first. When the agar plate was coagulated. the half amount war cut off and then the blood agar was took place in the space. On the border line, 5mm width, cotton threads which were contaminated with the larvae were placed. One hour after keeping at 15。C the number fo larvae which migrated to both sides were counted(Fig. 3).
Phototropism:Conical tubes: one was enamelled in black and the other was transparent, were used for experiment. A rectangular filter paper was put in each tube. The tubes were bridged with 0.5cm long filter paper where on the infective larvae were placed(Fig. 5). For another trial, 20cm long and 0.3cm diameter glass pipe was used. The pipe was enamelled in black in 5cm's turn. The larvae suspension was poured into the tube by a syringe and kept in 23。C temperature. Three hours after, the dark and light parts were cut by ampulecut and the larvae in each fraction were calculated(Fig.6).
Geotropis: In waxed paper box, 5cm height and 2.5cm width, silt, clay, sand soil, silt loam and clay soil was put respectively, During the experiment the soils were kept at moistured condition and in the dark place. The larvae suspension was placed in the middle portion of the soil. One th 5days after, the paper boxes were cut into 3 parts; upper, middle and lower and examined the number of the larvae in the soil samples(Fig. 7). In order to check the upward or downward migration of the
larvae, rectangular filter paper, 10cm long and 1cm width, was hung vertically, and the larvae suspension was smeared on the middle portion of the paper(Fig. 8). The edge of the paper was dipped in water; one at upper end and the other at lower end
and left at room temperature. One hour later, the paper were divided into four pieces from the top, upper, lower and bottom and calculated the number of larvae.
Histotropism: On the surfaces of the following rabbit organs; kidney, lung, liver, small intestine(outside) and skin(abdomainal portion), the larvae suspension was dropped and kept at room temperature. One hour later, the surfaces of each organ was washed, and the organs were put into Waring blendor. The larvae in the material were collected by Baermann's method for counting the larvae.
Comparison of skin and oral infection: For cutaneous infection, the abdominal wall of three mice were shaved without giving injury. Sterilized gauze pad was put on them and 1.0ml of larval suspension containing approximately 2,000 infective larvae were dampened on each gauze pad and the pads were removed after 2 hours and the pads were examined to calculate the remaining larvae on them. Orally, 1ml of larvae suspension was introduced into the stomach of the mouse through a stomach tube. Five days after the infection, the mice were sacrificed. The larvae in lung, liver, intestine and carcass were recovered by routine Baermann's method.
Resistance of the infective larvae in digestive juice: Two human gastric juices, pH 3.6 and pH 4.0 were used. Each juice was also divided into two groups according to temperature conditions; 37。C and 26。C. Larvae suspension was put into three conical tubes, containing 2ml of each group according to the pH and temperature, and the survivability of the larvae were examined at 4, 24 and 48 hours later. The resistance of the larvae was examined. also, in the artificial gastric juice(Pepsin 0.3gm, HCI conc. 0.7ml, and Aq. dist. 100.0pH 2.0) and intestinal juice(Sod.
bicarbonate 0.2gm. Trypsin 0.5gm, Aq. normal saline 100.1ml-pH 8.0). In this experiment, the distilled water was used as control.
Thermotropism: The recovery rate of the larvae of T. orientalis (T.O.) was 85.3% in 10。C tube and 14.7% in 35。C group, while the larvae of Ancylostoma caninum used in the control showed contrary phenomena: 5.1% in 10。C and 94.9% in 35。C tube.
Haemotropism: The larvae on the agar plate migrated to the blood-agar side more than to the simple agar plate. The balance showed high rate in 65.9% blood-agar side more than to the simple agar plate. The balance showed high rate in 65.9% blood-mixed than in 34.1% simple agar media.
Phototropism: In general, the larvae were found 60.4% from black-enamelled conical tube, but 39.6% from light side tube. In the enamelled glass pipe, the ratio between the dark and light was 71 : 29.
Geotropism and vertical migration: In the group which the upper edge dipped into water, the recovery rate was 43.8% in upper and 56% in lower part. In the group which the lower edge was dipped into water, the rater was 33.7% in upper part and 66.3% in lower part. The results show that the larvae can migrate up and down, but downwards movement is rather active than upward. In sandy soil the larvae showed negative geotropism, but there were no definite taxis in the silty and clay soil, probably due to the compact texture fo those soils.
Histotropism: The larvae were recovered 23.5% from kidney, 34.% from liver, 38.% from lung, 67.4% from intestine and 74.4% from skin.
Comparison of cutaneous and oral infection: By skin infection, totally 6.8% of the infected larvae were recovered from the mice and 6.3% by oral infection. The results indicate that the larvae are able th penetrate into skin or mucosal membrane by only chance.
Resistance in digestive juice: In human gastric juice, the larvae survived 100% at pH 3.6, 26。C for 48 hours, but at pH 4.0, at PH 4.0, the larvae survived 98% for 48 hours at 26。C and 55% at 37。C. In artificial gastric juice(pH 2.0), the larvae were destroyed 18% at 26。C , 48 hours and 28% at 37。C, on the same hours.
In intestinal juice(pH 8.0), the rates was 8% 26。Cand 98% at 37。C.
The biological natures of the infective stage larvae of Trichostrongylus orientalis were examined under various conditions. The followings are the summaries of the results:
1. The larvae showed negative thermotropism comparing to the hookworm larvae.
2. The larvae showed haemotropism in the blood agar plate.
3. The larvae showed negative phototaxis.
4. The larvae showed negative geotaxis in sandy soil.
5. The was no any phenomenon of geotaxis on the wet filter paper, but rather hydrotasix.
6. The larvae showed higher histotropism go skin than the other organs of mouse.
7. The was no difference on the migratory behaviour between oral infection and skin infection.
8. The larvae survived in digestive juices for 48 hours, but in intestinal juice the mortality rate was higher at 37 。C than at 26。C condition.