Pathways of pyrimidine nucleotide biosynthesis in gravid Angiostrongylus cantonensis.

The incorporation of radioactive precursors into pyrimidine nucleotides via de novo and salvage pathways was measured in gravid Angiostrongylus cantonensis by HPLC and thin-layer chromatography. 14C-labelled orotate, uridine, uracil and deoxyuridine were traced to UMP, UDP, UTP, UDP-glucose, dTMP, CMP, CDP and CTP. 3H-labelled cytidine was also incorporated into both uracil and cytosine nucleotides in a ratio of 2:1. Cytosine was a major end-product for all the precursors. Cytosine nucleotides were probably formed from UTP by the action of CTP synthetase whose activity in crude cell-free extract was 31.5 +/- 4.9 pmol min-1 (mg protein)-1. It was dependent on glutamine, ATP and GTP and was inhibited by CTP. The total amount of pyrimidine nucleotides formed from uridine was 3 times of that from uracil. The presence of uracil in the metabolism of uridine indicates that UMP is formed by uracil phosphoribosyltransferase as well as by uridine kinase. UMP is a key intermediate for cytidylate and thymidylate biosynthesis in the gravid worms.

Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.

Comparison between standard ELISA and dot-ELISA for serodiagnosis of human trichinosis.

The dot enzyme-linked immunosorbent assay (dot-ELISA) provided rapid and accurate detection of human trichinosis. Comparable sensitivity and specificity of the standard ELISA and dot-ELISA were obtained when sera of 18 confirmed cases of trichinosis were screened. Cross-reactivity was assayed with sera from 31 Chinese subjects with other parasitic infections. Only 2 cross-reactions occurred in the dot-ELISA, compared with 7 in the standard ELISA.

Prevalence of Toxoplasma gondii antibodies in the Chinese population of Hong Kong.

Sera from 2,499 randomly selected Chinese inhabitants of Hong Kong were tested for the presence of Toxoplasma gondii antibodies using the indirect fluorescent antibody test. The over-all prevalence was 9.8%. The prevalence was significantly higher in the 10 to 19 and 30 to 39 year age group and the highest level of titres was found in the 20 to 29 group. There was no significant difference in prevalence between the male and female population and between the rural and city dwellers. The distinctly low prevalence of toxoplasmosis in Hong Kong can probably be attributed to the absence of cats in most domestic households and the preference of the people to eat well-cooked meat (commonly beef and pork).

Study of acute trichinosis in Ghurkas: specificity and sensitivity of enzyme-linked immunosorbent assays for IgM and IgE antibodies to Trichinella larval antigens in diagnosis.

The enzyme-linked immunosorbent assay (ELISA) was successfully applied to detect IgE antibodies against parasitic antigens by using an additional antibody layer to attain an amplification effect. The sera of 18 Gurkha patients with clinical manifestations of acute trichinosis and 35 Chinese with other parasitic infections were tested for antibodies to Trichinella spiralis by IgE-, IgM- and IgG-ELISA, IgG-radioimmunoassay (RIA) and indirect haemagglutination test (IHA). ELISAs for detection of IgE and IgM antibodies provided a 100% specific and sensitive diagnosis. Although IHA, IgG-RIA and IgG-ELISA detected antibodies in 94% of patients, non-specific reactions were also observed in the two last named methods. Muscle biopsies were positive in only 56% of patients.

Four documented cases of eosinophilic meningoencephalitis due to Angiostrongylus cantonensis in Hong Kong.

4 cases of eosinophilic meningoencephalitis in Hong Kong are described. The major clinical features of the patients, who were 2-60 years old, were low grade fever, headache, mild meningeal signs, right facial palsy or hemiplegia. Eosinophilia in the peripheral blood and eosinophilic pleocytosis were prominent. Computerized tomography scans of brain showed a small area of attenuation with surrounding hypodense area; the lesion was resolved 1-2 months after admission to hospital. Electroencephalograms revealed abnormally slow dysrhythmia. Sections of a nematode observed in the brain of a patient who died were identified as those of young adult Angiostrongylus cantonensis. High ELISA titres against the crude antigens of this nematode were also noted in the serum of 3 patients. The disease is probably under-recognized in Hong Kong.

Further observations on the first documented outbreak of trichinosis in Hong Kong.

The first documented report of human trichinosis in Hong Kong is described comprising an outbreak amongst 20 Gurkha soldiers following a barbecue. The cardinal clinical features were fever, myalgia and facial oedema and the most useful laboratory tests were eosinophilia and elevated levels of creatinine phosphokinase. Gastrointestinal symptoms were uncommon. Seven patients who developed electrocardiographic abnormalities are the subject of an ongoing study. Four patients had psychiatric manifestations. Splinter haemorrhages, hypocalcaemia and evidence of renal dysfunction were absent. The parasite was recovered from 13 of patients and the diagnosis confirmed serologically in all. The value of the enzyme-linked immunosorbent assay (ELISA) for IgM and IgE antibodies is emphasized in that it is 100% specific and sensitive. Thiabendazole alone was used in treatment and all patients recovered.

Specificity of affinity-purified Trichinella spiralis antigens.

An affinity-purified fraction (APF) was obtained by passing crude somatic antigens of Trichinella spiralis muscle larvae through an Affi-Gel 10 column coupled with anti-Trichuris suis IgG. The fraction contained seven antigens with molecular weights ranging from 28 to 55 kDa. When tested with antiserum against other common nematodes of pigs from China, the APF was found to be markedly more specific than S3 antigens (prepared by a combination of cell fractionation and differential centrifugation according to Despommier and Lacetti, 1981) and fractions produced by Sephacryl S-200 gel filtration (F1 to F12). When the APF was used in an indirect IgG-enzyme-linked immunosorbent assay (IgG-ELISA) to screen serum samples from 2000 pigs imported from China, a positive rate of 7.5% was obtained. Similar screenings using the crude somatic antigens F1 and S3 gave a large number of cross-reactions and false positive reactions. Positive rates of 48%, 39% and 59.5% respectively were obtained for the three antigens.

Enhanced chemiluminescent enzyme immunoassay for the detection of trichinellosis antibodies in pigs.

A modified enhanced chemiluminescent enzyme assay (ECIA) was developed for mass screening of pigs for trichinellosis antibodies in abattoirs. Using Bionectics beads as solid support, the assay time could be reduced to 45 min. Optimal conditions for washing, blocking, incubation, concentration of serum, antigens and conjugates as well as timing of film exposure were determined. The sensitivity and specificity of the assay were found to be comparable to those of the triple antibody-IgG ELISA. The assay was tested in an abattoir and its efficacy was found to be satisfactory. However, the major disadvantage of the assay is the high cost of magnetic beads.

Purification of larval Taenia solium antigens by gel filtration.

Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.

Cross-reactions between crude antigens of larval Taenia solium (Cysticercus cellulosae) and other helminths of pigs.

A crude antigen extract of larval Taenia solium was shown by immunodiffusion (ID) and immunoelectrophoresis (IEP) to cross-react with rabbit antisera against pig serum proteins and larval T. hydatigena, and by enzyme-linked immunosorbent assay (ELISA) with antisera against pig serum proteins, Fasciolopsis buski, larval T. hydatigena, hydatid cyst, Hymenolepis diminuta and Dipylidium caninum. Immunoblotting demonstrated that the crude antigens extract contained epitopes of pig serum proteins of 48 and 66 kDa. The crude extract also contained a subunit of antigen B (95 kDa) which was also found in T. hydatigena and H. diminuta. Immunoperoxidase and indirect immunofluorescence studies showed that cross-reacting antigens were distributed mainly on the tegument of T. solium.

Purification of larval Taenia solium antigens by isoelectric focusing.

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3-8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)-ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.

Current status of food-borne parasitic zoonoses in Hong Kong.

Although there have been no recent epidemiological studies on parasitic zoonoses in Hong Kong, the following diseases are known to occur locally: toxoplasmosis, clonorchiasis, fasciolopsiasis, cysticercosis, hydatidosis, sparganosis, trichinellosis, angiostrongyliasis, gnathostomiasis and trichostrongylosis. Most of them are due to the consumption of infected animals imported from China. Due to the general unawareness of parasitic diseases by physicians and health authorities, the importance of zoonoses has been under recognized.

A brief update on the diagnosis of trichinellosis.

The latest immunological and molecular methods for the diagnosis of swine and human trichinellosis are briefly reviewed. The following topics are discussed in more detail: isolation of specific antigens by continuous elution-isoelectric focusing methods, production of recombinant antigens, nature of immunodominant antigens, potential use of heat shock proteins (HSPs) as diagnostic antigens, roles of specific IgE and circulating antigens (CA). The immunodominant antigens were found to be highly heat resistant. The specificity and sensitivity of colorimetric sandwich ELISA, microfluorescence (ELFA), enhanced chemiluminescence (ECIA) and dissociated enhanced lanthanide fluoroimmunoassay (DELFIA) in detecting CA were compared. The last method is the most sensitive, detecting as little as 1 ng of antigens/ml of serum. CA was detected as early as 7 days postinfection of mice. The serum from a patient suspected to have acute trichinellosis in Hong Kong was also tested positive for CA.

Production specific antigens from Trichinella spiralis using a continuous elution method and isoelectric focusing.

Immunodominant antigens of 45-53 kDa (one band per fraction) were obtained from excretory/secretory (E/S) and somatic products of infective-larvae of Trichinella spiralis using a continuous-elution method. They were further resolved by isoelectric focusing into different isoforms (45 kDa: pI4.47, 5.09, 5.47 and 5.86; 47 kDa: pI4.72 and 4.97; 53 kDa: pI4.86, 5.11, 5.44 and 5.78). In immunoblotting, the isoforms of pI 5.09, 5.86, 4.97, 5.44 and 5.78 did not cross-react with antisera against Trichuris suis, Metastrongylus apri, Gnathostoma hispidum and Stephanurus dentatus. Hence, they have the potential to serve as specific antigens for the serodiagnosis of trichinellosis.