Bile duct changes in rats reinfected with Clonorchis sinensis

This study describes an evaluation of the sonographic, cholangiographic, pathological, and immunological findings, and the protective effect shown by rats reinfected with Clonorchis sinensis. Eight experimental rat groups were, namely, a normal control, a primary infection control, a reinfection I (reinfection 7 week after treatment following 3-week infection), a reinfection II (reinfection 2 week after treatment following 8-week infection), a reinfection III (exploration of the intrahepatic bile ducts 1 week after reinfection 4 week after treatment following 4-week infection), a superinfection, a secondary infection control, and an infection following immunization group. Sonographic and cholangiographic findings showed moderate or marked dilatation of the bile duct confluence in the primary infection control, reinfection II, and secondary infection control groups. Juvenile worms survived in the intrahepatic bile ducts 1 week after reinfection following treatment in the reinfection III group. It was concluded that reinfecting juvenile worms found during the first week following reinfection failed to survive or grow further. Anatomical, pathophysiological, or immunological changes may induce protection from reinfection in rats.     

Susceptibility of experimental animals to reinfection with Clonorchis sinensis

The present study observed the resistance to reinfection with Clonorchis sinensis in various experimental animals including mice, guinea pigs, rabbits, and dogs, as well as rats and hamsters. The resistance rates to reinfection in rats, mice, hamsters, guinea pigs, rabbits, and dogs were 79.7%, 58.0%, -12.6%, 54.8%, 62.6%, and 6.0%, respectively. Worms recovered from reinfected rats and mice were immature, and significantly smaller than those from the primarily infected (P < 0.01), whereas those from other animals were fully matured to adults. These findings indicate that the protective response against reinfection with C. sinensis is prominent in rats and mice, and that they may be a good animal model to investigate the mechanism of resistance to reinfection with C. sinensis.     

Effects of gamma-irradiation on the infectivity and chromosome aberration of Clonorchis sinensis

Effects of gamma irradiation on the worm survival and chromosomal aberration of Clonorchis sinensis were studied. The metacercariae irradiated with various amounts of gamma radiation (ranging from 5 Gy to 50 Gy) were fed to rats, and the effects were compared with those of non-irradiated controls. Recovery rates of adult worms in irradiated groups were reduced gradually as increasing of the irradiation doses. No worm was recovered from rats which were fed with 50 Gy irradiated metacercariae. The chromosome number was 2n = 56 in all worms from all experimental groups. However, the groups irradiated with 20 Gy, 25 Gy or 30 Gy showed variations in the chromosome number, depending on different cells in the same individual. Radiation doses used in this study did not appear to induce chromosome aberrations, however, irradiation with 30 Gy showed slightly reduced chromosome size.     

Clonorchis sinensis metacercarial infection in the pond smelt Hypomesus olidus and the minnow Zacco platypus collected from the Soyang and Daechung Lakes

The pond smelt Hypomesus olidus and minnow Zacco platypus were collected from the Soyang and Daechung Lakes in January 2003, and their metacercarial infections was examined by the muscle compression and artificial digestion techniques. In the Soyang Lake, 161 metacercariae of Clonorchis sinensis (0.35 per fish) were harvested from 459 pond smelts examined. Also, 13 metacercariae of C. sinensis (0.43 per fish), 1 of Metagonimus sp., 4 of Echinostoma sp., 148 of Centrocestus armatus and 44 unidentified species were collected from 30 minnows. In the Daechung Lake, 369 metacercariae of C. sinensis (3.69 per fish) and 51 unidentified species were recovered from 100 pond smelts. The metacercariae of C. sinensis were fed to experimental rats, in which the adult flukes were identified. The pond smelts and minnows collected from the Soyang and Daechung Lakes were verified to be the second intermediate hosts and the sources of human C. sinensis infection.     

The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae

The effects of gamma irradiation on the survival and development of C. sinensis metacercariae were studied to evaluate the feasibility of irradiation as a control measure for clonorchiasis. Pseudorasbora parva were collected at an endemic river of clonorchiasis and were used for irradiation of the fluke in three schemes. The first (Scheme 1) was irradiation of the isolated metacercariae from the fish followed by infection to experimental rats. The second (Scheme 2) was irradiation of the fish, and then the metacercariae were isolated and infected to rats. The third (Scheme 3) was irradiation on the rat livers after infection with normal metacercariae. Irradiation doses varied from 5 to 100 Gy for Schemes 1 and 2, and 10 to 25 Gy for Scheme 3. The rats were sacrificed 2 to 6 weeks after infection. In Scheme 1, the metacercariae irradiated at 50 Gy failed to survive in the rats after 2 or 6 weeks. However, 1 to 44% of the metacercariae irradiated at 5-30 Gy survived. The estimated LD50 of Scheme 1 was 16.5 Gy. The flukes irradiated in Scheme 2 survived better than those in Scheme 1. The average worm recovery rate in 50 Gy was 28%(7-39% individually). Increasing the dose up to 100 Gy brought a remarkably low survival rate of an average 1%(0-3% individually). The LD50 of Scheme 2 was 47.5 Gy. Worm recovery rates in the 10 Gy group of Scheme 3 were 21-39%, and those in the 25 Gy group were 2% and 34%. Although the metacercariae were irradiated, all of the recovered worms were morphologically normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

ELISA of rat sera infected with Paragonimus iloktsuenensis

Enzyme-linked immunosorbent assay (ELISA) of paragonimiasis iloktsuenensis rat sera was performed using crude antigens of Paragonimus iloktsuenensis (PIA), P. westermani (PWA) and Clonorchis sinensis (CSA). Three crude antigens (PIA, PWA, CSA) were prepared to saline homogenated supernatants of whole adult worms. Infected rat sera were obtained biweekly from the albino rats fed 50-80 metacercariae of P. iloktsuenensis through gastric catheter. Experimental groups were divided into 4 groups: GI (controls), GII, GIII and GIV according to 1-7 worms as GII, 10-19 worms as GIII and 22-40 worms as GIV, respectively. In ELISA, the mean OD values of each group for the homologous antigen (PIA) were increased significantly compared to the control sera at the 4th week of infection. With the progress of duration of infection, the mean OD values of infected sera of GII & GIV continuously increased up to the 12th week (last week), but in GIII the mean OD value increased until the 10th week. No significance was noted among the infection dose groups (GII, GIII and GIV), after the 6th week of infection. Also, the OD values of all infected rats did not show any proportional relationships to the number of worms recovered. In brief, the antibody productivity of individual rats were strongly different. The rat sera infected with P. iloktsuenensis cross-reacted with those infected with P. westermani or C. sinensis, as identified by OD values.     

Changing patterns of serum and bile antibodies in re-infected rats with Clonorchis sinensis

Rats develop strong resistance to re-infection and super-infection by Clonorchis sinensis. The present study investigated the antibodies present in the sera and bile juice of rats that were primary infected and re-infected with C. sinensis. The serum level of specific IgG antibodies, which were elevated 2 wk of the primary infection, peaked at 4 wk and subsequently remained unchanged even during re-infection. The total IgE level in serum increased slowly from 388 ng / ml to 3,426 ng / ml beginning 2 wk after the primary infection, and remained high up to 8 wk but dropped to a normal level (259 ng / ml) after treatment. In resistant re-infected rats, the serum IgE level increased rapidly and peaked within 1 wk, whereas no increase was observed in immunosuppressed rats. The serum level of specific IgA antibodies was elevated beginning 1 wk after infection, and decreased 4 wk after treatment. The total bile IgA level unchanged during the primary infection but increased in treated and re-infected rats. The elevated levels of serum IgE and bile IgA indicate that these immunoglobulins may be correlated with the development of resistance to re-infection by C. sinensis in rats.     

Infection rates of Enterobius vermicularis and Clonorchis sinensis of primary school children in Hamyang-gun, Gyeongsangnam-do (province), Korea.

The egg positive rate of Enterobius vermicularis and Clonorchis sinensis of school children in the rural area was studied in Hamyang-gun, Gyeongsangnam-do in Korea. Cellotape anal swab and formalin ether concentration methods were performed one time to 720 primary school children. The total egg positive rate of E. vermicularis was 12.6% in two schools (Baekjeon and Wiseong). In the Baekjeon and Wiseong primary school, the egg positive rate of E. vermicularis was 4.6% and 13.4%, respectively. Pinworm egg positive rate was 17.6% in the lower grades (1st, 2nd and 3rd), and 7.7% in higher grades (4th, 5th and 6th). The total egg positive rate of male and female was 12.6% and 12.7%, respectively. The egg positive rate of C. sinensis of Baekjeon and Wiseong primary school was 1.5% and 0.46%, respectively. The total egg positive rate of C. sinesis was 0.56%. This survey showed that continuous education and chemotherapy is necessary to treat and prevent reinfection of E. vermicularis. In the case of C. sinensis, health education for school children is recommended to prevent potential infection of adolescents.     

Resistance to reinfection in rats induced by irradiated metacercariae of Clonorchis sinensis

A study was made to observe the association between the resistance to reinfection induced by irradiated metacercariae (MC) of Clonorchis sinensis and antigen specific Th1- and Th2-type cytokine productions in rats. Rats were infected with 20 MC of C. sinensis, previously exposed to a single dose of gamma irradiation, which varied from 0 to 100 Gy. All of them, single dose of 12 Gy showed higher IgG antibody titer with lowest worm recovery. Thus, 50 MC were used to challenge infection in rats previously infected with 20 MC irradiated at 12 Gy and the highest resistance to challenge infection was observed. The results of lymphocyte proliferation with specific antigen, ES Ag were shown no difference of proliferative responses as compared with primary and challenge infection at 12 Gy irradiation dose. In the case of cytokines production were observed that interferon (IFN-gamma) and interlukin (IL-2) were significantly enhanced, while IL-4 and IL-10 was almost unchanged to make comparison between primary and secondary infection at 12 Gy irradiation dose. In conclusion, the single dose of 12 Gy could be adopted for induction of the highest resistance to challenge infection. Up-regulation of Th1 type cytokines, IFN-gamma and IL-2 may be affected to develop vaccine by irradiated MC.     

Nippostrongylus brasiliensis: Increase of sialomucins reacting with anti-mucin monoclonal antibody HCM31 in rat small intestinal mucosa with primary infection and reinfection

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.     

Epidemiological study of clonorchiasis and metagonimiasis along the Geum-gang (River) in Okcheon-gun (county), Korea

The endemic status of clonorchiasis and metagonimiasis along the Geum-gang (River) in Okcheon-gun (County) in Korea was examined. From February to December 2000, stools of total 1,081 inhabitants living in 5 villages were examined. Each stool specimen was examined by both the cellophane thick smear method and the formalin-ether sedimentation technique. Egg-positive cases were further analyzed by Stoll's egg-counting technique, and praziquantel was administered to positive cases. The egg-positive rates for Clonorchis sinensis and Metagonimus species were 9.3% and 5.5%, respectively, and the double infection rate was 3.5%. The numbers of eggs per gram (EPG) of feces of C. sinensis and Metagonimus sp. were 918 +/- 1,463 and 711 +/- 947, respectively. The egg-positive rates for C. sinensis and Metagonimus sp. in the riverside area were 14.2% and 8.4%, respectively, which were significantly higher than those of the inland area (3.2% and 1.7%, respectively). The egg-positive rates of C. sinensis and Metagonimus sp. in males (16.7% and 10.0%) were significantly higher than those of females (3.5% and 1.8%). However, there were no significant differences of EPG values between localities and sexes. The prevalence of clonorchiasis and metagonimiasis in this survey was significantly lower than that in the previous reports. However, there is still a high prevalence of infection with C. sinensis and Metagonimus sp. in this region, especially in the riverside area.     

Infection status of Clonorchis sinensis in residents of Hamyang-gun,Gyeongsangnam-do,Korea

Oriental liver fluke (Clonorchis sinensis) infection was surveyed among residents of Hamyang-gun, Gyeongsangnam-do, Korea during the period of January 2001 to March 2002. Total 1,041 stool samples were collected from residents who visited Public Health Center and its branches in Hamyang-gun and examined using formalin-ether sedimentation method. The overall egg positive rate was 16%, male showing higher positive rate (21%) than female (10%). The age group of 30 to 50 years had the highest egg positive rate of C. sinensis from 20% to 22%. The positive examinees were treated with praziquantel and educated individually to prevent reinfection. Egg positive rate in this area was decreased when compared with results recorded in the past, however, still remained more than 10%. This study suggests that periodic examination, treatment as well as education of residents should be continued and systematized.     

不同毒力疟原虫感染早期根治性治疗对再感染体液免疫应答的影响

为探讨不同毒力疟原虫感染早期根治性治疗对再感染体液免疫应答的影响,用致死型和非致死型约氏疟原虫感染DBA/2小鼠,感染后3 d进行根治性治疗,并于初次感染后90 d进行再感染。计数红细胞感染率和检测再感染前（0 d）和再感染后（1、3、5 d）不同时间点脾细胞中活化性B细胞百分率以及血清中IgG、IgG1和IgG2 a水平,结果显示,再感染后2组小鼠均出现短暂的低水平虫体血症,再感染后第3天活化性B细胞、IgG、IgG1和IgG2 a均出现有意义的升高,但在每一相同检测时间点,2组小鼠的虫体血症、活化性B细胞百分率、IgG、IgG1和IgG2 a水平均没有显著差异。这些结果表明,不同毒力疟原虫感染早期的根治性治疗并不影响宿主在再感染时产生有效的体液免疫应答,强毒株原虫感染也能获得与弱毒株相同的可抵御再感染的能力。     

Collection of Clonorchis sinensis adult worms from infected humans after praziquantel treatment

A cohort was established for evaluation of cancer risk factors in Sancheong-gun, Gyeongsangnam-do, Korea. As one of the cohort studies, stools of 947 residents (403 males and 544 females, age range: 29-86 years) were screened for Clonorchis sinensis eggs using both Kato-Katz method and formalin-ether sedimentation technique. The overall egg positive rate of C. sinensis was 37.7% and individual EPG (eggs per gram of feces) counts ranged from 24 to 28,800. Eight egg positive residents voluntarily joined a process of collection of the passed worms after praziquantel treatment. A total of 158 worms were recovered from 5 of the 8 treated persons, ranged from 3 to 108 in each individual. The worms were 15-20 mm x 2-3 mm in size, and showed brown-pigmented, red, or white body colors. This is the first collection record of C. sinensis adult worms from humans through anthelmintic treatment and purgation. The adult worms of C. sinensis may be paralyzed by praziquantel and then discharged passively through bile flow in the bile duct and by peristaltic movement of the bowel.     

Curative and protective activity in rabbits after reinfection with Schistosoma mansoni: a new model of immunity?

New Zealand rabbits were infected on day 1 and challenged on days 15, 30, and 60 with 1,000 Schistosoma mansoni cercariae/animal/infection. Challenged and control rabbits were perfused 60 days after each infection, corresponding to days 75, 90, and 120 after the first exposure. No decrease in number of adult schistosomes occurred in animals reinfected 15 days after primary infection, but, when the rabbits were challenged 30 and 60 days after the first infection, worm burden reduction of 61.4% and 92.6%, respectively, was observed as compared to infection controls. These data indicate that rabbits submitted to reinfection are able to kill the worms from their primary infection, besides being protected against challenge parasites.     

High rate of Helicobacter pylori reinfection in children and adolescents

AIMS: Primary Helicobacter pylori infection occurs predominantly in childhood. The aims of this study were to establish the rate of H. pylori reinfection after successful eradication in children and adolescents and to determine the risk factors associated with reinfection. PATIENTS AND METHODS: This retrospective study involved 45 children (20 girls, 25 boys) who met the following criteria: eradication of H. pylori confirmed at least 4 weeks after the completion of therapy, and the search for reinfection at least one year after control of eradication of H. pylori. Demographic data, socioeconomic status and living conditions were recorded. RESULTS: Forty-five children aged 1.2-17.6 years (median, 10.9 years) at the time of H. pylori treatment were reviewed 1 to 9 years after H. pylori eradication. Eight children (18%) had been reinfected (5.4% to 6% per patient-year). Six of 25 (24%) children older than 10 years at the time of diagnosis became reinfected. None of the studied risk factors was associated with reinfection. However, having a sibling younger than 5 years was found in four of seven (57%) reinfected children versus five of 24 (21%) nonreinfected children (p = .08). CONCLUSION: Children become reinfected more frequently than adults. Adolescents become reinfected, whereas acquisition of primary H. pylori infection occurs predominantly in early childhood. Close contact with young children, especially siblings, younger than 5 years could be a more important risk factor than the age of the patient at the time of treatment for the high rate of reinfection in childhood.     

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

Corbicula fluminea (Bivalvia: Corbiculidae): a possible second molluscan intermediate host of Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea.

More than 1,500 clams of Corbicula fluminea, the most favorable food source of freshwater bivalves in Korea, were collected from 5 localities to examine cercarial and metacercarial infection with Echinostoma cinetorchis. Although 3 clams infected with suspicious E. cinetorchis metacercariae out of 200 specimens collected at Kangjin, Chollanam-do were detected, no cercarial and metacercarial infections with E. cinetorchis were observed in field-collected Corbicula specimens. In the susceptibility experiments with laboratory-reared clams, those infected with miracidia of E. cinetorchis did not release their cercariae up to 60 days after infection. To confirm the identity of second intermediate host of E. cinetorchis experimentally, a total of 30 clams were exposed to the cercariae from Segmentina hemisphaerula that had been infected with miracidia of E. cinetorchis. The clams were susceptible to cercariae of E. cinetorchis with an infection rate of 93.3%. Metacercariae from clams taken more than 7 days after cercarial exposure were fed to rats (S/D strain), and adult worms of E. cinetorchis, characterized by 37-38 collar spines on the head crown, were recovered from the ileocecal regions. This is the first report of C. fluminea as a possible second intermediate host of E. cinetorchis.     

Influence of primary infection on the population dynamics of Nematospiroides dubius after challenge infections in mice

Dobson C., Sitepu P. and Brindley P. J. 1985. Influence of primary infection on the population dynamics of Nematospiroides dubius after challenge infections in mice. International Journal for Parasitology15: 353–359. Similar proportions of the inoculum of Nematospiroides dubius larvae reached sexual maturity by 14 days after administration of 50–400 larvae but more adult worms had been expelled by day 63 after infection from those mice infected with 50 vs 400 larvae. There was a significant correlation between time and worm expulsion for all inoculum size groups except for mice given 400 larvae.In mice reinfected with 100 larvae, after termination of primary infections derived from 10 through 400 larvae, more worms from the challenging dose were recovered from mice given greater compared with those given smaller numbers of larvae at primary infection. The N. dubius population size after challenge infection was correlated positively both with number of larvae administered as the primary infection and with the resultant population size during that infection. The serum anti-N. dubius antibody titres after reinfection were higher in mice given 400 compared with those given fewer larvae at primary infection, and the fecundity and female to male sex ratio of the N. dubius populations decreased in proportion to these antibody titres.Protective immunity against challenge N. dubius infection, in mice which had been drenched free of adult worms established from 400 larvae for 5 down to 1 weeks before reinfection, increased from 45% (1 week) to 80% (5 weeks). There was a negative correlation between the population size of N. dubius during challenge infection and the duration between anthelmintic treatment and challenge infection.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.