Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection.

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.

Cloning, expression, and characterization of iron-containing superoxide dismutase from Neospora caninum.

A gene encoding superoxide dismutase (SOD) from Neospora caninum, a causative agent of neosporosis, has been cloned and its gene product functionally expressed and characterized. The gene had an open reading frame of 606 bp and deduced 201 amino acids. Sequence analysis showed that the gene had conserved metal-binding residues and conserved amino acid residues that were found in Fe-SODs. Comparison of the deduced amino acid sequence of the enzyme with previously reported Fe-SOD amino acid sequences of the other parasitic protozoans revealed significant high homology. The coding region of the N. caninum Fe-SOD was cloned and functionally expressed in Escherichia coli. Enzyme activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide and potassium cyanide, and the enzyme showed similar biochemical properties with typical Fe-SODs of other parasitic protozoans. Southern blot analysis showed that the SOD gene appears to be present as a single-copy gene in N. caninum genome. Semiquantitative reverse transcription-polymerase chain reaction and immunoblot using antiserum raised against the purified recombinant protein showed that Fe-SOD is expressed in both developmental stages of N. caninum, i.e., in bradyzoites and tachyzoites. In an immunofluorescence assay, the enzyme was localized on the cell surface of N. caninum tachyzoites. These results suggest that Fe-SOD might be essential for the intracellular survival of N. caninum and may play an important role in the pathogenesis of the parasite by protecting the parasite from oxidative killing.

Purification and characterization of a 43-kilodalton extracellular serine proteinase from Cryptococcus neoformans.

An extracellular proteinase was purified from culture filtrates of Cryptococcus neoformans NHPY24 by DEAE ion-exchange chromatography and gelatin affinity column chromatography with azoalbumin as the substrate. The molecular mass of the purified enzyme was 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its pH optimum was 7.0 to 8.0, and maximal activity was obtained at pH 7.5 and 37 degrees C. By isoelectric focusing, the purified enzyme had a pI of 4.77. Enzyme activity was inhibited by serine proteinase inhibitors such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. The purified enzyme was thus a serine proteinase. It hydrolyzed natural substrates including hemoglobin, beta-casein, and gamma globulin.

Identification of enteroviruses by using monoclonal antibodies against a putative common epitope.

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.

Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis.

A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5' and 3' regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.

Degradation of immunoglobulins, protease inhibitors and interleukin-1 by a secretory proteinase of Acanthamoeba castellanii.

The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (sIgA), IgG, and IgM. It also degraded interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Its activity was not inhibited by endogenous protease inhibitors, such as alpha 2-macroglobulin, alpha 1-trypsin inhibitor, and alpha 2-antiplasmin. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthamoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection.

Effect of anthelmintics on the early stage of Enterobius vermicularis.

In order to determine the susceptible age of Enterobius vermicularis to anthelmintics and to observe the chronologic growth of female E. vermicularis in man, experimental infections were done. About 500 eggs were challenged to 19 volunteers. After 4, 8, 16, 20, 24, 28, 32 and 35 days of infection, each case was treated by either mebendazole or pyrantel pamoate. On the 40th day of infection all cases including control were treated again to terminate the expermental infection and to evaluate the effect of previous treatment. Each case collected 3-day stools to harvest the expelled worms. The results could be summarized as follows: The infection rates of females were in range of 0.6~13.1% in control cases. Because the collected worms showed comparable growth and development by day, the worns were concluded to be derived from experimental infection. Cases that were treated with mebendazole on 4, 8 and 16 days after infection expelled 37.5%, 2.5% and 67.5% of the number expelled by a control case on the 40th day. Cases treated thereafter expelled no worms on the 40 days. Cases that were treated with pyrantel pamoates on 4, 8, 16, 24, 28, 32 and 35 days, expelled 90.7%, 25%, 45.3%, 8%, 2.7%, 5% and 29.3% of the number collected from control cases in respect. All the worms collected were females. The total body length increased consistently and comparably from the 20th day of infection. Those collected on the 20th day were 2.5~3.0 mm long with vigina, sac-like structure and strands of ovaries; 24 day-old worms may have short uterus, 28 day-old worms had long uterus without eggs, 32 day-old worms began to produce eggs, 35 day-old worms showed wide variations in egg deposit in uterus, and 40 day-old worms had uterus filled with eggs from vulva to anal levels. From the above results, it was inferred that the life span of female Enterobius vermicularis was longer than 40 days, and the developmental stages of worms younger than 16 days resisted considerably to both mebendazole and pyrantel pamoate.

[Studies on the intestinal fluke, Metagonimus yokogawai Katsurada, 1912 in Korea Geographical distribution of sweetfish and infection status with Metagonimus metacercaria in south-eastern area of Korea]

The present survey was undertaken to observe the geographical distribution of metacercaria among the sweetfish, Plecoglossus altivelis, the most important second intermediate host of Metagonimus yokogawai, which were collected during the period from 1983 to 1984 in south-eastern coastal areas of Korea. The results obtained were summarized as follows: 1. A total of 668 sweetfish collected from 70 chons (=streams) in the above coasta1 areas was examined for the metacercariae, and 432(64.7 percent) among them were infected with metacercariae of M. yokogawai. The mean number of metacercariae in a fish was in the range from 0 to 29,604 and the mean number of the metacercariae in a fish was 640.3. 2. It was observed that the mean infection rate showed 46.4 percent in Gangweon-Do (=province). The infection rates varied from stream to stream showing 100 percent in Namdae stream(Gangnun-city), Obsib-stream(Samchok-gun) and Dongmak, 95.0 percent in Hosan stream, 90.0 percent in Gungchon stream, and Namdae stream (Yangyang-up), 80.0 percent in Hwasang stream, 50.0 percent in Daebong stream, 45.0 percent in Yonghwa stream, 30.0 percent in Sangchon stream and Sachon stream, 20.0 percent in Munam stream and Okgyoe stream, 15.0 percent in Imweon stream, 10.0 percent in Namchon stream. And no metacercarial infections in their fish host were found in the areas of Mulchi stream, Sangun stream, Gosamun stream, Jonchon stream snd Gagok stream. 3. It was observed that the mean infection rate showed 50.9 percent in Kyongsangbuk-Do(=province). The infection rates also varied from stream to stream showing 100 percent in Songchon stream, Osib stream (Yongdok-gun) and Daejong stream, 60.0 percent in Pyonghae stream snd Gunmu stream, 50.0 percent in Buhwung stream, 25.0 percent in Nagok stream, 20.0 percent in Oangbi stream, 10.0 percent in Namhodong stream. And no metacercarial infections in their fish host were found in the areas of Namdae stream (Uljinup) and Choksan stream. 4. It was observed that the mean infection rate showed 67.6 percent in Kyongsangnam-Do(=province). The infection rates varied from stream to stream showing 100 percent in Taehwa river, Hwiya river, Inchon stream, Miryang river, Nam river, Hwagae stream, Koha stream, Sosang stream, and Tongchon stream, 90.9 percent in Yoncho stream, 90.0 percent in Hoihwa stream, 80.0 percent in Chindong stream, 66.7 percent in Chingyo stream, 40.0 percent in Hoyam river. And no metacercarial infections in their fish host were found in the areas of Chinjon stream, Sanchon stream and Jukchon stream. 5. It was observed that the mean infection rate showed 94.6 percent in Chollanam-Do(=province). The infection rates varied from stream to stream showing 100 percent in Dongchon stream, Isachon stream, Somjin river (Goksong-gun), Somjin river (Kurye-gun), Sosi stream, Gokchon stream, Kohung stream, Kwansan stream, Youi stream, Unjon stream and Apnae stream, 92.3 percent in Tamjin river, 90.9 percent in Okkok stream, 84.6 percent in Songgun stream. And no metacercarial infections in their fish host were found in the area of Yongok stream. 6. On the other hand, the infection rate showed 100 percent in Inchon river of Chollabuk-Do (=province) and Kangon stream of Cheju-Do (=province).

[Aspartate And Alanine Aminotransferase In Fasciola Hepatica]

The activity and distribution of aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in adult Fasciola hepatica have been studied. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of GOT and GPT was measured by the method of Reitman and Frankel. Isozyme patterns of those enzyme were also examined by DEAE-cellulose column chromatography. The results obtained were as follows: 1. The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1 g of Fasciola hepatica, respectively. 2. The activity of those enzymes was relatively low compared with those in mammalian tissues. 3. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71 % of the activity was in cytosolic, 24 % in mitochondrial and 5 % was in nuclear fraction. 4. About 22 % of the total alanine aminotransferase activity was found in the mitochondrial fraction, about 66 percent in the cytosolic fraction. 5. Aspartate aminotransferase from cytosolic fraction was separated into two types of isozymes, whereas alanine aminotransferase from cytosolic fraction gave only one active peak on DEAE-cellulose column chromatography.

[Purification And Properties Of Branched Chain Amino Acid Aminotransferase From Fasciola Hepatica]

The distribution and properties of branched chain amino acid aminotransferase(EC 2.6.1.42) was investigated in adult Fasciola hepatica. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of branched chain amino acid aminotransferase was measured by the method of Ichihara and Koyama (1966). Isozyme patterns of this enzyme was also examined by DEAE-cellulose column chromatography. The results obtained were as follows: 1. The activity in homogenate was found to be 12.69 units/g wet tissue. The activity of this enzyme was relatively high compared with those in rat tissues. 2. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8 % of the activity was in cytosolic, 10.9 % in mitochondrial and 1.3 % was in nuclear fraction. 3. Cytosolic fraction of Fasciola hepatica contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase. Enzyme I was eluted by 50 mM phosphate buffer from DEAE-cellulose column and catalyzed the transamination of all three branched chain amino acids. 4. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. 5. The best substrate among three amino acids (leucine, isoleucine and valine) was L-isoleucine. 6. The optimal temperature of Enzyme I was 45 degrees C and the optimal pH was 8.2. 7. The Km value for leucine of Enzyme I was 4.17 mM. 8. The Km values for alpha-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41 mM and 4.76 x 10(-3) mM, respectively.

Morphological Observation Of Enterobius Vermicularis Expelled By Various Anthelmintics.

When enterobiasis cases were treated with anthelmintics only for one time, the interval to recurrence was variable by different drugs used. And this phenomenon is supposedly connected with biological or developmental cycle of the worm and the consequent efficacy of the different anthelmintics. This study was undertaken to confirm this fact by studying the expelled worms morphologically to correlate the anthelmintics efficacy and stage of worm development in Enterobius vermicularis. A total of 131 children in 3 orphanages was examined by 4 anal swabs (mean positive rate, 80%). They were randomized into 5 experimental groups. Each group was treated with placebo, mebendazole, pyrantel, pyrvinium, and piperazine (70 mg/kg, single dose) respectively. After treatment, all stool were collected for 3 days to get the expelled Enterobius. A total of 6,165 pinworms was studied under the microscope. The sex was discriminated and the length was individually measured. A number of male pinworms was collected in all groups. Females of 2~11 mm in length were also collected in 5 groups. However, significantly larger number of short females was observed in mebendazole group compared with other groups. Twenty-one days after the first treatment, all children were again treated with mebendazole. Once more stool were examined. A total of 1,853 worms was collected. In mebendazole group, there were no females longer than 8.74 mm in the second treatment. In pyrvinium group, 8.31mm in lenght was the longest for female. However in control, pyrantel and piperazine groups, females of 2~11 mm in length were collected. From above results, one could conclude that the removing ability of mebendazole and pyrvinium was satisfactory for the worms in the early stage of development in Enterobius. Pyrantel and single dose of piperazine showed less effective in worm reduction ability especially on those at the early stages.

[Effects Of Prednisolone Injection On The Liver Of The Mouse Inoculated With The Adult Worms Of Clonorchis Sinensis Intraperitoneally]

In order to understand the effect of prednisolone injection on the histopathological changes of the mouse liver and the chronological changes of the worm structure of Clonorchis sinensis, when this fluke was inoculated to the mouse intraperitoneally. The recovery rate, survival rate, location and size of the inoculated worms as well as the histopathological changes of the liver were investigated for the comparison among the groups of mice, which were classified by number of worms and the duration of observation period. The result obtained were summarized as follows. 1. The recovery rate and survival rate of the worms decreased especially 28 days after the inoculation. 2. Most of worms (45.5%) were collected from the peritoneal cavity, and some of worms were found tightly adherent to the capsules of the liver, spleen, intestine and diaphragm. 3. The mean worm size after inoculation was constantly smaller than that before inoculation. 4. Remarkable atrophy in the reproductive organs of the worm, such as spermatheca, testes, vitelline gland and ovary was frequently observed at the 10th day of inoculation. 5. Histopathologically the liver failed to show any parasitic worm inside the intrahepatic biliary system. However, multiple well formed egg-containing granulomas were present along the liver capsule. These necrotic granulomas were occasionally found under the fibrotic liver capsule. Focal necrosis and focal phlebitis together with vascular dilatation were prominent features of the liver. 6. The worms recovered in the capsule of the liver were degenerated and necrotized. Usually, there were remarkable capsulitis and granuloma formation around the eggs.

[Study On The Chromosomal Proteins Of Fasciola Hepatica]

In attempt to investigate histone fractions and non-histones of parasites, nuclei were isolated from Fasciola hepatica by the procedure of Pogo et al. (1966). Histone fractions H1, H2a, H2b, H3 and H4 were prepared from isolated nuclei by the procedure of Johns (1964 and l967). The five histone fractions found in most tissues were also present in the Fasciola hepatica histones. These histone fractions were characterized by amino acid analysis and by polyacrylamide disc gel electrophoresis. Non-histone proteins were extracted from isolated Fasciola hepatica nuclei and separated by SDS-polyacrylamide gel electrophoresis. The results of the experiment were summarized as follows: 1. The yield of whole histone recovered was 2.47 mg per 1 g of Fasciola hepatica. 2. The yield of DNA was 1.02 mg per gm of tissues. Consequently the DNA to histone ratio was 1:2.44. 3. The relative amounts of five fractions, i.e., Hl, H2a, H2b, H3 and H4 were 19.96%, 26.48%, 29.60%, 12.56% and 14.37%, respectively. 4. Amino acid analysis of the individual histone fractions showed that the over-all compositions were similar but not identical to those of corresponding fraction from calf thymus. 5. It was found that histone H2b fraction of Fasciola hepatica contained detectable amounts of epsilon-N-monomethyllysine. No evidence for the presence of methylated lysine or other side-chain derivatives was reported on this histone fraction. 6. In SDS-polyacrylamide disc gel, it showed that 17 protein bands of nuclear acidic protein can be identified visually.

[An Experimental Study On The Mouse Intraperitoneal Inoculation Of The Adult Worm Of Clonorchis Sinensis]

The present experimental study was undertaken to observe the chronological change of the worm structure of Clonorchis sinensis and the pathological findings of the liver when this fluke was inoculated to the mouse intraperitoneally. The recovery rate, survival rate, location and size of the inoculated worms as well as the pathological changes of the liver were investigated for the comparison among the groups of mice, classified by number of worms and the duration of experiment. The results obtained were summarized as follows: 1. The recovery and survival rates of the worms decreased especially 28 days after the inoculation. 2. Most of worms (90.l percent) were collected from the peritoneal cavity and some of worms were found tightly adherent to the capsules of the liver, spleen, stomach, intestine and diaphragm. There were no worms recovered penetrated in the parenchymes of these organs. 3. The mean worm size after inoculation was smaller than that before inoculation. At the 10th day after the inoculation, the shrinkage of posterior portion of the worm body was observed. 4. Remarkable atrophy in the reproductive organs of the worm, such as spermatheca, testes, vitelline glands and ovary was frequently observed at the 10th day of inoculation. 5. Histopathologically the liver failed to show any parasitic worm inside the intrahepatic biliary system. However, multiple well formed egg-containing granulomas were present along the liver capsule. These necrotic granulomas were occasionally found under the fibrotic liver capsule. Focal necrosis and focal phlebitis together with vascular dilatation were prominent features seen in the liver. The bile duct in the liver showed mild dilation of the lumen, flattening of epithelial cells and periductal small round cell infiltration. Neither adenomatous hyperplasia nor portal fibrosis was seen in the whole experimental groups. Foci of intralobular micro-granulomas were found in some experimental animals. 6. The worms recovered in the capsule of the liver were degenerated and necrotized. Usually, there were remarkable capsulitis and granuloma formation around the eggs.

[A study on the lipids of Chinese liverfluke, Clonorchis sinensis]

The present study was undertaken to observe the quality and quantity of lipids in the adult worms of Chinese liverfluke, Clonorchis sinensis. Lipid extraction was done by the methods of Folch et a1. (l957) and Kenny (1952), and then the extracted lipid fractions of the worm were separated by thin layer chromatography. Those fractions were also subjected to perform the quantitative analyses of glycerides, cholesterols and phospholipids. The results obtained were summarized as follows: 1. Total amount of glyceride was 37.56 mg per gram of worm tissue and the amount of monoglyceride was 8.34 mg per gm; diglyceride, 15.46 mg per gm; and triglyceride, 12.86 mg per gm. 2. Total amount of cholesterol was 3.30 mg per gm of worm tissue, and the esterified cholesterol (1.72 mg/gm) was a little more than that of free cholesterol (1.26 mg/gm). 3. The following 8 phospholipids were detected in the worm tissue of C. sinensis, i.e., lysophosphatidylcholine, phosphatidylcholine, phophatidylinositol, sphingomyelin, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and an unknown phospholipid.

[Anthelmintic Effect Of Oxantel And Oxantel/pyrantel Tablets Against Intestinal Nematode Infections]

Present trial was carried out to evaluate the anthelmintic efficacy of oxantel/pyrantel tablets against intestinal nematode infections, and to determine the efficacy of oxantel tablels against Trichuris infection. A total of 34 subjects with the mixed infections were treated with oxantel/pyrantel tablets (100 mg/tablet each) in a single dose of 15 mg/kg body weight, and another group of 22 Trichuris infected cases received oxantel pamoate tablets (125 mg/tablet) in a single dose of 15 mg/kg. All stool examinations were done before the treatment and 3 weeks after the treatment. The cellophane thick smear (Kato's technique) and Stoll's dilution egg counting method were employed. The results of the trial were summarized as follows: 1. The cure rates (egg negative conversion rates) and egg reduction rates for oxantel/pyrantel tablets were 85.3% and 97% in trichuriasis, 100% each in ascariasis and ancylostomiasis. 2. The oxantel tablet treated group demonstrated a cure rate of 90.9% and an egg reduction rate of 96.3% in the treatment of Trichuris. 3. There were no detectable objective and subjective side effects in this trial. Both oxantel/pyrantel and oxantel pamoate tablets were readily accepted and well tolerated.

[A Study On The Lipids Of Ascaris Suum Ova]

The present study was undertaken to observe the quality and quantity of lipid constituents by the developing Ascaris suum eggs. The collected eggs from the uterus of A. suum were classified into 3 groups, i.e., single cell stage, morula stage and embryonated eggs, and were subjected to analyse their lipid fractions. To obtain the morula stage eggs, 10 to 11 incubation days at 30 degree C were needed and for the embryonated eggs, 30 to 31 days were lasted. At the time of experiment, their indices of development by Hoffman were 285(morula stage) and 42 (embryonated stage) respectively. Lipid extraction was done by the methods of Folch et a1. (1957) and Kenny (1952), and then the extracted lipid fractions from the above 3 groups of eggs were separated by thin layer chromatography. Those fractions were also subjected to perform the quantitative analyses of fatty acids, glycerides, cholesterol and phospholipids. The results obtained were summarized as follows. 1. Total amount of fatty acid was decreased from 12.9 mg per gram of eggs (single cell stage) to 6.6 mg/gm (embryonated eggs), whereas the proportion of free fatty acid to total fatty acid was constantly increased from 77.5% to 89.4% during the period of egg development. 2. Total amount of glycerides was also increased from 33.0 mg/gm of single cell stage to 55.9 mg/gm of the embryonated eggs. The most abundant glyceride among 3 glycerides discovered from A. suum eggs was triglyceride, and the least was monoglyceride. 3. The amount of free cholesterol was much larger than that of ester form in general, and it reached maximum in the eggs of morula stage (4.6 mg/gm). The increase of total cholesterol was monitored during the development of A. suum eggs from 3.3 mg/gm to 5.4 mg/gm. 4. The following 8 phospholipids were detected in the embryonated eggs, i.e., lysophosphatidyl choline, phosphatidyl inositol, sphingomyelin, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl ethanolamine and unknown phospholipid. But in the single cell stage eggs, 4 kinds out of the above 8 phopholipids were not observed, and in the morula stage eggs, 2 kinds were absent among the 8 phospholipids.

[Studies On The Lungfluke, Paragonimus Iloktsuenensis: VI. Effect Of Prednisolone Injection On The Immune Responses Of Albino Rats]

In order to understand the effect of prednisolone injection on the immune responses of albino rat against Paragonimus iloktsuenensis infection, the differential leucocyte counts, appearance of immunoblast (large pyroninophilic cell, LPC) in the spleen and lungs in various experimental groups were observed in relation with the growth, maturation and migration sites of this rodent lungfluke. Rats of 3 experimental groups (A series), each group consisted of 5 rats, were infected with 20 metacercariae of P. iloktsuenensis, and they were kept for 3 days(Group I), 3 weeks(Group II), and 4 weeks (Group III) of infection period. The same number of experimental groups, each group of rats received 10 mg/kg dose of prednisolone injection every other day, were also kept and examined in comparison with the former groups. Preparation of peripheral blood smear and collection of worms were completed immediately after the end of infection period, and they were stained with Giemsa or Semichon's acetocarmine. Paraffin sections of the spleen and the lung tissues were stained with hematoxylin-eosin and methyl green pyronin (MGP). Those materials from A and B series of experimental groups were examined under the light microscope, and the results obtained were as follows: 1. On observing differential leucocyte counts of peripheral blood smear, lymphocyte counts were consistently higher than those of uninfected controls in A series of infected groups, while those of B series were consistently low. On the other hand, neutrophil counts of A series showed lower counts than those of B series. In general, fluctuation patterns of both A and B series of experimental groups were almost the same, although lymphocyte and neutrophil counts showed reciprocal relation. The eosinophil counts of both series were negligible, especially in the groups of B series. 2. The counts of LPC in the periarterial lymphatic sheath of the spleen were rapidly increased in the groups of A series, while those of B series were much less than those of A series, and the appearance of considerable LPC in the spleen was also delayed in B series. Furthermore, LPC of peribronchial lymphatic tissue in A series started to increase after the invasion of lungflukes into the lungs, while those of B series were much less due to the inhibited migration of lymphocytes into the lesions. 3. Number, size and maturity of collected worms showed no significant differences between the groups of A and B series, but migration speed of the lungflukes was somewhat accelerated in B series than in A series. In this connection, it was considered that the immune responses of albino rats did not contribute for the complete protection against P. iloktsuenensis, but inhibited the migration of this lungfluke to some extent.