Topology of feather melanocyte progenitor niche allows complex pigment patterns to emerge.

Color patterns of bird plumage affect animal behavior and speciation. Diverse patterns are present in different species and within the individual. Here, we study the cellular and molecular basis of feather pigment pattern formation. Melanocyte progenitors are distributed as a horizontal ring in the proximal follicle, sending melanocytes vertically up into the epithelial cylinder, which gradually emerges as feathers grow. Different pigment patterns form by modulating the presence, arrangement, or differentiation of melanocytes. A layer of peripheral pulp further regulates pigmentation via patterned agouti expression. Lifetime feather cyclic regeneration resets pigment patterns for physiological needs. Thus, the evolution of stem cell niche topology allows complex pigment patterning through combinatorial co-option of simple regulatory mechanisms.

The infectivity and antigenicity of Toxocara canis eggs can be retained after long-term preservation.

Suspensions of fertilized eggs of Toxocara canis were mixed with 2% neutral formalin and preserved at 4 degrees C. When, after storage for 0, 12, 18, 21 and 24 months, samples of the eggs were incubated at 30 degrees C for 12 days, 96.8%, 92.6%, 74.1%, 51.0% and 19.3% of the eggs in the samples were found to embryonate. The embryonated eggs produced from the fertilized eggs preserved (in 2% neutral formalin at 4 degrees C) for 0, 12, 18 and 21 months were then tested for their infectivity to BALB/c mice, each mouse being given 800 embryonated eggs. The numbers of larvae recovered from the mice and the sites from which they were recovered, 2 or 14 days post-infection, appeared unaffected by the length of storage of the eggs. The infected mice all had similar eosinophil counts in their peripheral blood and similar serum titres of Toxocara-specific IgM and IgG antibodies, and cultures of their spleen cells produced similar amounts of interleukin-4, interleukin-5 and interferon-gamma when stimulated with concanavalin A. The results of SDS-PAGE indicated that egg preservation for at least 21 months had no effect on the excretory-secretory antigens in samples of medium from cultures of infective larvae released from the eggs. In summary, at least 50% of the fertilized eggs preserved in 2% neutral formalin at 4 degrees C for 21 months could fully embryonate and then had the same infectivity and antigenicity as embryonated fresh eggs.

Hepatic Schistosomiasis japonica in a patient with gallstones and bile duct stones--a case report.

Schistosomiasis, a common parasitic disease in many countries, is found as imported cases in Taiwan. Responsible for human infections are five species, one of which, Schistosoma japonicum, is currently endemic in China and South-east Asia. Chronic infection with S. japonicum may lead to the development of liver fibrosis, calcification and portal hypertension. Under investigation by sonography and computed tomography (CT) scan, a peculiar "turtle-back" appearance of liver fibrosis and calcification may be found. Herein, we report a case referred to our department due to jaundice. The sonography of liver showed typical "turtle-back" appearance. Gallstones and bile duct stones were also found in this case. Surgical interventions with percutaneous transhepatic biliary drainage (PTBD), cholecystectomy and choledocholithotomy were performed to relieve the obstructive jaundice and remove the stones. There were no parasitic eggs in the extracted stones or in drained bile juice. However, deposits of calcified S. japonicum eggs in liver parenchyma and portal tracts were identified in liver biopsy. No special treatment was given for the schistosomiasis japonica because the calcified parasitic eggs were the sequelae of past infection.

Eosinophilic meningitis caused by Angiostrongylus cantonensis: report of 17 cases.

PURPOSE: To describe two outbreaks of Angiostrongylus cantonensis infection that occurred in Kaohsiung, Taiwan, during 1998 and 1999, and to characterize the source of the outbreaks and the clinical manifestations of the disease. SUBJECTS AND METHODS: We performed a retrospective cohort study among Thai laborers with eosinophilic meningitis who ate raw snails (Ampullarium canaliculatus), as well as an environmental surveillance of larvae in snails. RESULTS: We enrolled 17 Thai laborers in whom severe headache and eosinophilia developed within 4 to 23 days after eating raw snails. Twelve (71%) developed eosinophilic meningitis. Third-stage larvae were found in the cerebrospinal fluids of 2 patients and in all 12 tested snails. Specific antibodies to A. cantonensis were detected in serum from 16 of the patients and in cerebrospinal fluid from 5 of the patients. Central nervous system manifestations included headache (n = 17 [100%]), fever (n = 11 [65%]), Brudzinski's sign/stiff neck (n = 11 [65%]), hyperesthesia (n = 3 [18%]), cranial nerve palsy (n = 2 [12%]), diplopia (n = 2 [12%]), and ataxia (n = 1 [6%]). Laboratory findings included peripheral eosinophilia (n = 15 [88%]) and cerebrospinal fluid eosinophilia (n = 12 [71%]); elevated immunoglobulin (Ig) E levels (n = 13 [100%]); and transient increases in white blood cell count (n = 7 [41%]) and in serum levels of creatine kinase (n = 7 [41%]), transaminase (n = 3 [18%]), and lactate dehydrogenase (n = 2 [12%]). The severity of illness and eosinophilia were correlated with the number of ingested snails. Meningeal and basal ganglion enhancement was noted on magnetic resonance imaging in several patients. Treatment with mebendazole combined with glucocorticosteroids appeared to shorten the course of the infection, but not the number of relapses. The eosinophil count fell to normal within 3 months, but IgE levels remained elevated for as long as 6 months. All patients recovered with minimal neurologic sequelae. CONCLUSION: Eosinophilic meningitis caused by A. cantonensis should be considered in patients who have headache or central nervous system manifestations after eating raw snails.

An outbreak of meningitis caused by Angiostrongylus cantonensis in Kaohsiung.

Eight Thai laborers developed meningitis after eating raw snails (Ampullarium canaliculatus) during the period from September 27 to October 6, 1998. The diagnosis of Angiostrongylus cantonensis infection was established in all patients by serologic studies of serum and cerebral spinal fluid (CSF). Clinical manifestations included meningitis, radiculitis and cranial nerve palsy. Symptoms included fever, headache, orbital pain, gastrointestinal upset, hyperesthesia, muscle weakness, skin rash and diplopia. Laboratory abnormalities included peripheral eosinophilia, CSF eosinophilia, transient elevation of liver enzymes and creatinine phosphokinase, elevation of IgE. No space occupying lesions were detected by magnetic resonance imaging of the brain. None of the patients developed severe sequelae during the 6-month follow-up except for occasional headache in one patient. This report also provides evidence that third stage larvae were present in the intermediate host, A. canaliculatus, which the laborers had eaten.

Immunodiagnosis of human eosinophilic meningitis using an antigen of Angiostrongylus cantonensis L5 with molecular weight 204 kD.

An antigen from Angiostrongylus cantonensis fifth-stage larvae was purified by immuno-affinity chromatography with a specific monoclonal antibody. The purified antigen showed only a single band with a molecular weight of 204 kD in SDS-PAGE, and no cross-reactivity to antibodies induced by several other species of helminths were observed in ELISA. When the purified antigen was used to examine serum and cerebrospinal fluid (CSF) specimens by ELISA, the antibody levels in patients with eosinophilic meningitis or meningoencephalitis (EME) were significantly higher than those of control subjects. The antibody levels in serum were slightly higher than those in CSF, and the levels in serum were positively correlated with the levels in CSF. The reliability in detection of antibodies in serum was slightly higher than that in the detection of antibodies in CSF specimens. The purification of a specific A. cantonensis antigen and its subsequent use in the development of an ELISA for detection of A. cantonensis specific antibodies in serum specimens constitute an important step towards improvement in the accuracy of diagnosis for A. cantonensis infections.

A survey on the intestinal parasites of the school children in Kaohsiung county.

The present study on the prevalence of intestinal parasites infection, from September to December 1999, was conducted among school children in Taoyuan Hsiang, Kaohsiung county. The investigated areas included three (Jiannshan, Shingjong and Taoyuan) primary schools. The overall infection rate in 305 children determined by Merthiolate-Iodine-Formaldehyde Concentration method of stools was 17%. Four confirmed species of helminthes (Ascaris lumbricoides, Hookworm, Trichuris trichiura and Hymenolepis nana) and three species of protozoa (Giardia lamblia, Entamoeba coli and Blastocystis hominis) were detected. Males and females had the infection rates of 24% and 11%. The infection rates of aboriginal and non-aboriginal children were 17% and 14%, respectively. Grade 1 and Grade 6 had the highest infection rate (21%). Following tape perianal examination of 302 children, the overall infection rate of Enterobius vermicularis was 25%. Males and females had the infection rates of 24% and 26%. The infection rates of aboriginal and non-aboriginal children were 27% and 11%, respectively. Grade 1 had the highest infection rate (37%). Based on these data, the infection rate of intestinal parasites among rural primary school children in Kaohsiung county remains high.

Improvements in the infectivity of cryopreserved third-stage larvae of Angiostrongylus cantonensis using a programmable freezer.

Although there have been some advances in the cryopreservation of Angiostrongylus cantonensis, the degrees of viability and infectivity of the cryopreserved developmental stages have not been high. A two-step freezing protocol using a programmable freezer was determined to be effective in improving the infectivity of the cryopreserved third-stage larvae of this parasite. After washing steps and suspension in 10% (v/v) dimethylsulfoxide and equilibrium at room temperature the larvae were placed into the freezer. The temperature was lowered first at 0.8 degrees C/min from room temperature to -40 degrees C and then at 10 degrees C/min to -70 degrees C. The samples were plunged into liquid nitrogen. After storage in liquid nitrogen for 7-15 days the larvae were thawed rapidly in 37 degrees C water and 27.6% were found to show vigorous "S-shape" movement without significant changes in appearance. These larvae (50/rodent) could develop to the fifth stage in mice (42.6%) and establish patent infection in rats (40.4%). Moreover, there was no significant difference in the recovery rates of cryopreserved worms and their unfrozen counterparts. These findings indicate that steady precooling conditions may decrease damage with regard to the infectivity of cryptopreserved third-stage larvae of A. cantonensis.

Humoral immune responses in mice infected with gamma-irradiated third-stage larvae of Angiostrongylus cantonensis.

Humoral immune responses in C57BL/6 strain mice infected with 1.0 kGy gamma-irradiated third-stage larvae (L3) and with non-gamma-irradiated L3 of Angiostrongylus cantonensis were assessed in this study. The young-adult worm (L5) antigen-specific antibodies in sera of both groups of mice elevated gradually and reached a peak in the third week after infection. The IgM antibodies of mice 1-week after infection with gamma-irradiated L3 and with non-gamma-irradiated L3 recognized several L5 antigens of the same molecular weight. However, only partially identical profiles were observed in the reaction of L5 antigens with IgM and IgG antibodies from mice 3 weeks after infection with gamma-irradiated L3 and with non-gamma-irradiated L3 respectively by western blot analysis. With regard to antibody-dependent cell-mediated cytotoxicity, antibodies from 3-week infected mice significantly increased the adherence of mouse eosinophils and neutrophils to L3 of A. cantonensis. The immunoresponses of splenic cells from mice in the third week of infection to sheep red blood cells were suppressed because the number of plaque forming cells obviously decreased more than the uninfected control.

Detection of circulating antigen by monoclonal antibodies for immunodiagnosis of angiostrongyliasis.

Two monoclonal antibodies, which recognize the epitope on an antigen with a molecular weight of 204 kD from the fifth-stage worm of Angiostrongylus cantonensis, were previously prepared and used to detect circulating antigens in patients with eosinophilic meningitis or meningoencephalitis and in mice experimentally infected with this parasite by a double-antibody, sandwich enzyme-linked immunosorbent assay (ELISA). The levels of this circulating antigen in experimentally infected mice were significantly higher three weeks after infection. The ELISA values in the detection of circulating antigens in cerebrospinal fluid (CSF) from patients were markedly higher than those in serum. Immunodiagnosis of patients with angiostrongyliasis by this technique proved to be highly specific for circulating antigens in serum and CSF specimens; however, the sensitivity in CSF was significantly higher than in serum.

Role of T cell subpopulations in mice infected with Angiostrongylus cantonensis.

When C57BL/6 mice were infected with Angiostrongylus cantonensis, the percentage of T helper (CD4+) cells and T supressor (CD8+) cells in peripheral blood increased weekly until the third and seventh week respectively, and then gradually decreased. C57BL/6 mice were depleted of CD4+ and CD8+ T cells by in vivo injection of anti-CD4 and anti-CD8 monoclonal antibodies, respectively, and then infected with A. cantonensis. There were significantly more and less worms recovered in the mice depleted of CD4+ and CD8+ T cells respectively than in undepleted mice. Discrete subpopulations of T cells from mice exposed to A. cantonensis for 3 weeks or 7 weeks were adoptively transferred to syngeneic recipients which were then given a challenge infection. Protection was mediated by a CD4+ T cell population present in mice after 3 weeks of infection but was not demonstrable with cells taken 7 weeks after infection. When CD4+ T cells obtained from 3-week infected mice were mixed with 5% CD8+ T cells obtained from mice infected for 7 weeks, no significant transfer of resistance was observed. Thus, immune responses to A. cantonensis in mice were regulated by discrete subpopulations of T lymphocytes.

[Parasitic infections among wild rats from two areas of Kaohsiung].

Wild rats including four strains were captured for examination of parasitic infections. The numbers were 101 from areas of Kaohsiung harbor and 98 from areas of Kaohsiung airport. Most of the examined wild rats were Rattus norvegicus and second most were Suncus murinus. The infection rates of intestinal helminths in rats of both areas did not display any significant difference. The overall infection rate was 29.6%. The infection rate was highest in R. norvegicus and the most frequently found parasite in rats was Hymenolepis diminuta. Moniliformis moniliformis and Angiostrongylus cantonensis were revealed in two separated R. norvegicus rats. The infection rates of intestinal helminths in R. norvegicus were significantly correlated with the size of rats. There was no find in microscopic observation of blood smear from these rats stained with Giemsa's dye.

The susceptibility of five stains mice to infections with Angiostrongylus cantonensis.

The susceptibility to the infection of Angiostrongylus cantonensis was studied in five inbred stains of mice including four H-2 haplotypes. Three weeks after infection, C57 BL/10J (H-2b) and C57BL/6J (H-2b) stains of mice showed the lowest, CBA (H-2k) and A/J (H-2a) stains showed intermediate, and the BALB/cJ (H-2d) mice showed the greatest susceptibility to infection. BALB/cJ mice also displayed the most body weight loss, the highest number of worms and showed the least number of days of survival. The s susceptibility of C57BL/6J (H-2b) and C57BL/10J (H-2b) strains of mice sharing same H-2 haplotype was the same despite their different genetic backgrounds. These data suggested that the H-2 gene might influence the susceptibility of mice to A. cantonensis.

[Investigation on the parasites of alien laborers in Kaohsiung].

In the past year, stool specimens of 1,732 alien laborers were examined for intestinal parasites. There was no significant difference in the positive rate between males and females. All alien laborers were examined in 3 hospitals and the positive rates were 24.8% (191/769), 13.6% (109/804) and 12.6% (20/159). Broken down by nationality, the positive rates in laborers from Thailand, Indonesia, the Philippines and Malaysia were 23.9% (192/803), 20.3% (62/305), 12.1% (60/498) and 4.8% (6/126) respectively. Of all alien laborers, 17.2% (297/1,732) had single infections of parasites and 1.3% (23/1,732) had multiple infections. Opisthorchis viverrinii was the most prevalent parasite found in infected alien laborers. 91.5% of alien laborers were aged from 21 to 40-years-old and had positive rates of parasites reaching 91.0%. The time of arrival in Taiwan had an effect in all alien laborers examined for parasites. The positive rate in laborers arriving during April to June was 22.7% which was significantly higher than the 16.3% for laborers arriving in other months.

Antibodies in serum of patients with clonorchiasis before and after treatment.

Sera of 31 patients infected with Clonorchis sinensis were examined using fraction 1 antigen by ELISA during a one-year observation. The results of ELISA with Igs, IgG and IgA demonstrated high sensitivity (100%, 100% and 90%) and specificity (100%, 100% and 87%). Sera specific Igs and IgG were significantly decreased in the 3rd month after treatment with praziquantel (25mg/kg body weight in one dose), and IgA was significantly decreased in the 1st month (paired t-test, p < 0.05). No eggs were found in the stool after treatment. Detection of sera specific Igs, IgG and IgA by ELISA was combined with stool examination to evaluate the effect of praziquantel and the completeness of the cure.

Antibodies of different immunoglobulin isotypes in serum and bile of patients with clonorchiasis.

Specimens of serum and bile from patients were assayed by an enzyme-linked immunosorbent assay to reveal antibodies specific to antigens from adult Clonorchis sinensis. Antibodies of the IgG isotype showed the greatest elevation during infection (Student's t-test, P < 0.001), whereas serum IgA and IgE and secretory IgA in bile were moderately elevated (P < 0.05). Major differences in the distribution of antibodies among the IgG subclasses were observed between patients who were and those who were not infected. IgG4 antibody levels were elevated in the serum and bile of infected patients, and there were significant correlations between the levels of IgG and IgG4 antibodies and the intensity of infection.

Detection of antibodies to Angiostrongylus cantonensis in serum and cerebrospinal fluid of patients with eosinophilic meningitis.

Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student's t-test, P less than 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF.

Characterization of monoclonal antibodies to young-adult worms of Angiostrongylus cantonensis.

Three IgG1 and one IgG2a monoclonal antibodies were obtained by fusion of NS-1 cells with spleen cells of BALB/C mice immunized with young-adult worm antigen of A. cantonensis. These 4 monoclonal antibodies were specific to A. cantonensis without cross-reactivity to any antigens of Toxocara canis, Ascaris suum, Clonorchis sinensis, Dirofilaria immitis, Paragonimus westermani, Gnathostoma spinigerum, Strongyloides stercoralis and Anisakis spp. detected by ELISA. The affinity constants of monoclonal antibodies specific to young-adult worms antigen of A. cantonensis were in the range of 10(7) M-1 to 10(9) M-1. The immunoblots showed that the 4 monoclonal antibodies recognized epitope on molecular weight of 204 kiloDaltons. They will be utilized to purify their corresponding antigen by an immunoaffinity column for the specific immunodiagnosis.

A survey of Ampullarium canaliculatus for natural infection of Angiostrongylus cantonensis in south Taiwan.

Ampullarium canaliculatus were collected monthly from the field in five areas in Pintung County and examined for larvae of Angiostrongylus cantonensis. Snails from one area showed the highest positive rate (30.8%) and the highest number (31) of larvae per snail. Snails were placed in four groups according to size. The positive rates in snails increased from 10.0 to 29.4% and the average number of larvae per snail increased from 11 to 40 as the size of snails increased from 1.0 to 4.0 cm height or more. In the seasonal fluctuation of snails in the field, positive snails could be examined every month; however, the positive rate in snails (28.2%) and the average number of larvae per snail (33) in the dry season (November to April) were significantly higher than those (15.2% and 23 respectively) in the rainy season (May to October). Ampullarium canaliculatus were confirmed naturally infected with A. cantonensis in this investigation so more attention should be paid to the snails to prevent the infection in humans.