John S. Welch

Immunodiagnosis of parasitic zoonoses: comparative efficacy of three immunofluorescence tests using antigens purified by affinity chromatography.

Three immunofluorescent antibody assays were developed during this study: the direct (DFAT) and indirect (IFAT) fluorescent antibody tests using frozen worm sections, and the cyanogen bromide indirect fluorescent antibody test (CNBr-IFAT) using helminth antigens purified by affinity chromatography bound on to commercially prepared CNBr-Sepharose 4B beads. Purified antigens used in the CNBr-IFAT gave greater specificity and sensitivity than either the DFAT or IFAT. Dirofilaria immitis, Toxocara canis, Angiostrongylus cantonensis and Ascaris lumbricoides infections were studied in natural and zoonotic hosts and in rabbits immunized with antigens prepared from these parasites. In addition, a serological survey of Aboriginal Australians from Queensland was made.

Immunodiagnosis and seroepidemiology of Angiostrongylus cantonensis zoonoses in man.

The development of serum and cellular assays to measure responses to Angiostrongylus cantonensis antigen, purified by affinity chromatography, formed the basis of this study. The specificity and sensitivity of these techniques were established in immunized rabbits and in naturally and laboratory-infected rats. The assays were then used to determine levels of immunological responsiveness to A. cantonensis in four Australian populations. There was a direct correlation between the prevalence of the parasite in rodents and the proportion of human reactors to A. cantonensis antigen in each population studied. Five patients with similar clinical histories and presenting symptoms suggesting eosinophilic meningitis were investigated; three were admitted to hospital. Haematological examination demonstrated hypereosinophilia in all five while three had, in addition, a cerebrospinal fluid eosinophilia. Serological tests and assays of cell-mediated responses to A. cantonensis antigen showed elevated immunological reactivity during the acute phase of illness with a subsequent decrease in reactivity corresponding with the progressive recovery of the patient.

Immunodiagnosis of parasitic zoonoses: Purification of Toxocara canis antigens by affinity chromatography

Abstract A method of affinity chromatography developed for the purification of species-specific antigens from Toxocara canis adult worms is described. Immunochemical analyses by polyacrylamide gel electrophoresis, immunoelectrophoresis and immunodiffusion showed that ‘pure’ antigen contained fewer but more specific proteins than ‘crude’ antigen. Purified antigens and parasite sections from four parasite species ( Toxocara canis, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris lumbricoides ) were used in immunofluorescence tests to measure serum antibody levels in animals with natural or experimental T. canis infections and people with zoonotic toxocariasis. ‘Pure’ antigen showed higher specificity and sensitivity than ‘crude’ antigen in serological testing.

CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small‐cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty‐nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA‐D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti‐p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti‐p14ARF antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour‐suppressor genes mapping both centromeric and telomeric of this locus.

Immunodiagnosis of Entamoeba histolytica and Ascaris lumbricoides infections in Caucasian and Aboriginal Australians

The immunodiagnostic efficiency of an indirect immunofluorescence test (IFAT) and in vitro lymphocyte proliferative responsiveness (cell mediated immunity test, CMIT) used to measure the immunological responses of individuals with known natural Entamoeba histolytica and Ascaris lumbricoides infections, was studied under survey conditions. E. histolytica was common among Aborigines from Cherbourg, Kowanyama and Central Australia, but it was not found in Brisbane Caucasians. The protozoan was selected for the study because it was prevalent and purified antigen was commercially available. Immunodiagnosis for A. lumbricoides was made using an antigen prepared by affinity chromatography. Diagnosis based on frequency distribution of immunological data gave valid assessment of the number of infected individuals in each population studied.

Angiostrongylus cantonensis in East New Britain, Papua New Guinea

The first case of angiostrongyliasis (eosinophilic meningitis) to be diagnosed in Papua New Guinea in 1977, led to a survey of adult Melanesian residents of East New Britain province where the case occurred, to assess the level of immunological responsiveness to Angiostrongylus cantonensis. In 104 subjects tested using an indirect fluorescent antibody test, the serum antibody titres to A. cantonensis equalled or exceeded 1:16 in 50.7% and ranged from 1:32 to 1:64 in 17.3% of subjects. No individual tested showed evidence of active or recent angiostrongyliasis but in a patient suspected to have ocular angiostrongyliasis, the antibody titre of A. cantonensis was 1:128 in the single serum specimen obtained. In a programme to determine the distribution of this parasite in the natural hosts, 19 rats were trapped; one had adult A. cantonensis in its pulmonary arteries. Examination of 200 Achatina fulica snails revealed Angiostrongylus larvae in 62 (31%) and when these larvae were fed to laboratory rats, adult A. cantonensis were subsequently recovered from their pulmonary arteries.

Immunodiagnosis of parasitic zoonoses: sensitivity and specificity of in vitro lymphocyte proliferative responsiveness using nematode antigens purified by affinity chromatography

The sensitivity of in vitro lymphocyte proliferative responsiveness using antigens purified by affinity chromatography, was greater than that of three serological tests and allowed the specific identification of Dirofilaria immitis, Toxocara canis, Angiostrongylus cantonensis and Ascaris lumbricoides in animals sensitized with parasite antigens, and the diagnosis of natural (D. immitis, T. canis and Angiostrongylus cantonensis) and zoonotic nematode infections. Lymphocytes remained viable in whole blood suspensions in RPM 1640 in a polystyrene insulation box at air temperature for at least 12 hours and reacted in the cell-mediated immunity test (CMIT) without loss of response. The CMIT proved useful in the early immunodiagnosis of nematode infections and showed that parasite antigens purified by affinity chromatography retained a high degree of specificity and sensitivity in both the CMIT and serological tests.