Study on the prevalence of Toxoplasma gondii and Neospora caninum and molecular evidence of Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis infections in red foxes (Vulpes vulpes) in rural Ireland.

Thoracic fluid (pleural fluid and clotted blood) from 206 foxes were examined for antibodies to Toxoplasma gondii and 220 thoracic fluid samples were tested for Neospora caninum antibodies using indirect immunofluorescent antibody tests (IFAT). A total of 115 (56%) and six (3%) foxes had antibodies to T. gondii and N. caninum, respectively. The brains from 148 foxes were examined for histological lesions and pathological changes suggestive of parasitic encephalitis were observed in 33 (22%). Two thirds of these foxes had antibodies to T. gondii and one fox had antibodies to both T. gondii and N. caninum. PCR assays carried out on DNA extracted from the 33 brains with histological lesions were negative for N. caninum but one of the brains was positive for T. gondii. Microsporidian DNA was also amplified from the brains of two of these foxes. Sequencing these amplicons revealed 100% homology with Encephalitozoon (Septata) intestinalis in one fox and Encephalitozoon cuniculi in the second fox. This is the first report of Encephalitozoon infections in wildlife in Ireland.

Purification of egg yolk immunoglobulins. A two-step procedure using hydrophobic interaction chromatography and gel filtration.

A two-step chromatographic procedure was developed for the isolation and purification of hen IgY antibodies from egg yolk. The antibodies were completely separated from vitellin and lipids by hydrophobic interaction chromatography followed by gel filtration. Almost no residual yolk proteins, no immunoglobulin aggregates, and no antibody fragments could be detected in the final extract. Moreover, the method described, guarantees the recovery of antibodies of undiminished activity. Although the final yield is somewhat lower than that obtained by an isolation method consisting of two precipitation steps with polyethylene glycol and alcohol respectively, the procedure described is particularly recommended when highly purified antibody preparations are needed.

Occurrence of Cryptosporidium sp. oocysts in fecal and water samples in Austria.

Oocysts of Cryptosporidium spp. were detected and differentiated by a modular arranged gene amplification procedure in various samples, mostly human stool, feces of herpetotaxa, and water, in different locations of South and Eastern Austria. Cryptosporidium parvum was found in stool samples of immunocompromised persons, in reptile feces, and in water samples. The presence of Cryptosporidium in an area is probably associated with high human population densities since water from protected sources in sparsely inhabited areas is rarely contaminated.

Microbiological irrigation water quality of the Marchfeld canal system.

The Marchfeld basin with a size of approximately 1000 km2 represents the Austrian "granary". To prevent shortage of water as a result of increased ground water removal for irrigation, industrial purposes and drinking water supply, a canal being 18 km in length was constructed from the Danube to the center of the Marchfeld. From there, water is further distributed via two creeks (Russbach and Stempfelbach). This study was intended to evaluate whether the surface water of the Marchfeld canal system can be classified into hygienic-microbiological categories as proposed by DIN (Deutsche Industrienorm) standards for irrigation water. For this purpose, water sampled monthly from three different sampling sites from 1996 to 1999 was examined for E. coli and enterococci. In addition, water samples were examined for salmonella twice a year from 1996 to 1998 and for cryptosporidia six times during the year 1999. Though the water showed varying degrees of fecal load, the results of the examinations revealed that only one of the three sampling sites showed constant quality levels according to the DIN classification system over prolonged periods of time. However, exceeding of the limit values was occasionally observed indicating the need for regular bacteriological examinations. The high variation of the results from the other sampling sites hardly permits a definite classification in one of the quality classes.

Diagnosis of Pneumocystis carinii pneumonia by bronchoalveolar lavage in AIDS patients. Comparison of Diff-Quik, fungifluor stain, direct immunofluorescence test and polymerase chain reaction.

OBJECTIVE: To assess the sensitivity, specificity and accuracy of Diff-Quik, fungifluor stain, the direct immunofluorescence test (DIFT) and the polymerase chain reaction (PCR) in the diagnosis of Pneumocystis carinii pneumonia (PCP) in human immunodeficiency virus (HIV)-infected patients. STUDY DESIGN: From December 1992 through November 1993, 112 bronchoalveolar lavage fluid (BALF) samples were obtained from 80 HIV-infected patients. BALF samples were processed for cytologic and microbiologic analysis and for PCR. Cytologic examination was carried out on Diff-Quik-stained cytocentrifuge preparations and with May-Grünwald-Giemsa staining and fungifluor staining. For diagnosis of PC infection, DIFT and PCR were used. RESULTS: Thirty-two of 112 acute episodes were caused by P carinii. Diff-Quik had the highest sensitivity (84.8%) as compared to fungifluor stain (60.0%), DIFT (59.4%) and PCR (65.6%). The specificity was 98.7% with Diff-Quik, 100% with fungifluor stain, and 98.6% and 97.3% with DIFT and PCR, respectively. Accuracy was high with every method (94.4% with Diff-Quik, 88.3% with fungifluor stain, 86.7% with DIFT and 87.6% with PCR). CONCLUSION: Diff-Quik is a good diagnostic tool in the diagnosis of PCP. The combination of Diff-Quik and fungifluor stain is recommended because of its cost-effectiveness and because of its rapid diagnosis of severe PCP. PCR and DIFT should be used only on patients judged clinically to have PCP with discrepant results in Diff-Quik and fungifluor stain in BALF samples.

[Differential diagnosis of ascites and abscess forming hepatitis in AIDS patients with reference to the first patient with microsporidia infection in Austria]. / Zur Differentialdiagnose des Aszites und der abszedierenden Hepatitis bei AIDS-Patienten anhand des ersten Patienten mit Mikrosporidieninfektion in Osterreich.

We report on a 30 years-old female AIDS patient suffering from generalized pneumocystosis and intestinal microsporidiosis. The chest X-ray showed a right-sided pleural effusion; the lungs showed no areas of consolidation and the heart and the vessels were normal in size. Sonography revealed multiple cystic lesions of the liver measuring 1-3 cm in diameter, as well as ascites. Pneumocystis carinii was detected on cytological examination of the bronchoalveolar lavage fluid, the pleural effusion and the ascitic fluid. Intestinal microsporidiosis was diagnosed by cytological examination of the stool. Both pneumocystis carinii infection and microsporidiosis may cause hepatitis and ascites. In our patient the organ manifestations of the two infections did not overlap. Since simultaneous organ manifestations are possible the differential diagnosis is discussed. This is the first case of microsporidiosis reported in Austria.

[Diagnosis of pneumocystis carinii pneumonia in AIDS patients]. / Zur Diagnostik der Pneumocystis carinii-Pneumonie bei AIDS-Patienten.

BACKGROUND: Based on the changing disease pattern of human immunodeficiency virus (HIV) associated pulmonary complications we conducted a prospective study in order to compare the value of laboratory tests in patients with Pneumocystis (P.) carinii pneumonia (PCP) and other pulmonary complications and of different identification methods of P. carinii in bronchoalveolar lavage fluid (BALF) in PCP patients. PATIENTS AND METHODS: In 217 HIV-1-infected patients we evaluated the following parameters: platelets, serum lactat dehydrogenase (LDH), total serum protein (TP), hemoglobin (Hb), and CD4+ and CD8+ T-lymphocyte count. P. carinii was identified in BALF by May Grünwald Giemsa stain (MGG), direct immunofluorescence test (DIFT), and polymerase chain reaction (PCR). We correlated these parameters in patients with a presumptive diagnosis of PCP and compared them with those of patients suffering from other pulmonary complications. RESULTS: All patients underwent bronchoscopy. 55 patients (25.3%) had a presumptive diagnosis of PCP. The sensitivity values of MGG stain, DIFT, and PCR differed considerably (79.1%, 56.1%, and 65.9%, respectively), but specificity values did not (99.2%, 97.3%, and 98.2%, respectively) as well as accuracy values (93.8%, 86.2%, and 89.7%, respectively). The mean values of platelets, of LDH, and of total serum protein of PCP patients and those of patients with other pulmonary diseases differed statistically significant as well as the mean values of these parameters of PCP patients and those of patients with bacterial pneumonia. Logistic-regression analysis revealed the number of platelets and the amount of total serum protein as independent, significant prognostic factors. Moreover, each PCP patient had a CD4+ T-lymphocyte count of less than 200 cells/mm3 blood. The CD4/CD8 ratio of PCP patients was statistically significant lower than that of patients with bacterial pneumonia. CONCLUSIONS: A detection of P. carinii in BALF is inevitable for a definitive diagnosis of a PCP. The most efficient identification method in this case is the MGG stain. Platelets, total serum protein, and CD4+ T-lymphocyte count should be included into the criteria for the presumptive diagnosis of PCP.

A rapid and simple method of purification of Toxoplasma gondii trophozoites originating from tissue culture for use in the indirect immunofluorescent antibody test.

A simple and convenient purification method for Toxoplasma trophozoites from tissue culture cells by density centrifugation is described. Separation of cells treated with formaldehyde is achieved by means of a Percoll solution with a density of 1.056 g/ml permitting removal of more than 99% of the cells and cell debris from the tissue culture. Thus highly purified trophozoites of Toxoplasma gondii raised in tissue cultures become available for serological tests, particularly for the indirect immunofluorescent antibody test.

Antigens of Toxoplasma gondii recognized by sera of AIDS patients before, during, and after clinically important infections.

A longitudinal study of different parameters of the immune responses to Toxoplasma gondii was performed with sera of AIDS patients taken during and after clinically important Toxoplasma infections. Follow-up of patients lasted for 9 months on an average. The titres of the specific IgG and IgM antibodies were measured by an indirect fluorescent antibody test (IFAT), and the appearance of circulating antigens of Toxoplasma gondii was determined in 88 sera of 18 patients with CNS (6 cases), pulmonary (1), lymph-node toxoplasmosis (1), or asymptomatic primary infections (2), respectively. The profile of the IgG antibodies reacting with a lytic antigen originating from a pool of trophozoites of six different Toxoplasma strains were examined by means of an SDS-PAGE followed by an immunoblot. Although numerous antigen bands were recognized by the sera of patients with clinically important infections, an antigen pattern characteristic of an acute infection could not be discovered. The majority of these sera, however, recognized bands at 27 and 57 kd; proteins of these molecular weights are components of the circulating antigens. In patients without any indication of a Toxoplasma infection, small amounts of antibodies reacting with 34-38 kd antigens were detected. The results of this study demonstrate that seropositivity to Toxoplasma gondii in AIDS patients determined by routine serological methods (e.g. IFAT) may be very heterogeneous even if identical titres are found; it simply results from different combinations of various antibodies which can only be detected by the immunoblotting technique.

Detection and characterization of circulating antigens in acute experimental infections of mice with four different strains of Toxoplasma gondii.

Acute infections with four different strains of Toxoplasma gondii, all of them being highly pathogenic for mice, were provoked by intraperitoneal injection of 2.2 x 10(7) trophozoites. The times of the appearance of circulating antigens in the sera of the infected mice were determined and the amounts of these antigens were measured by an enzyme-linked immunosorbent assay. The molecular weight of the circulating antigens was determined by gel filtration and by Western blot following PAGE. The isoelectric points of these antigens were determined by immunoblotting after isoelectric focusing. Circulating antigens were detectable up to ng amounts/ml serum from the 1st day p.i. onwards. The circulating antigens consisted of a number of proteins with molecular weights of greater than 10(4), 300, 65, 25, and less than 5 kd. The isoelectric points of these proteins were situated between pH 3.5 and pH 7. Time of appearance and structure of the circulating antigens were very similar in all four Toxoplasma strains. Circulating antigens are apparently generated regularly in experimental acute Toxoplasma infections, and lysis of parasite cells appears to be the major mechanism of their formation.

Parasitic infections in HIV patients in Austria: first results of a long-term study.

In the course of a long-term study of parasitic infections among HIV-infected persons in Austria during the period from November 1985 until May 1989, 618 persons infected with HIV (including 270 hospitalized patients, most of them with severe symptoms of AIDS) were examined. 58% of all persons had antibodies against Toxoplasma gondii. The incidence of clinically overt toxoplasmosis was about 20% in the 167 hospitalized persons infected with the parasite. In 29% of 68 patients with suspected pneumocystosis, the infection could be verified. In 9% of 219 patients, Cryptosporidium sp. was found. In two persons, an infection with Strongyloides stercoralis was diagnosed. Except these AIDS-associated opportunistic infections, the incidence of parasitic infections in the Austrian HIV-infected population was found to be low, and, except for Entamoeba histolytica, not significantly exceeding the frequency of parasitic infections in non-HIV-infected Austrians. Compared to data on the frequency of opportunistic infections in AIDS-patients in other developed countries, toxoplasmosis as well as infections with Cryptosporidium sp. seem to be more often diagnosed in Austria, whereas pneumocystosis is slightly less frequently found.

Evidence of structural proteins of Toxoplasma gondii in sera of experimentally infected mice.

The present study was performed to clarify whether structural proteins are constituants of the antigens of Toxoplasma gondii which circulate in the sera of experimentally infected mice. Rabbits were immunized with mice sera containing circulating antigen, the rabbit sera were then tested for antibodies against Toxoplasma gondii. Low titers of specific antibodies, directed against cell wall proteins, could be detected. Thus, the circulating antigen must at least partially consist of structural proteins.

Isoenzyme studies on Toxoplasma gondii isolates using isoelectric focusing.

Zymogram analysis using isoelectric focusing on polyacrylamide gels was performed to characterize and distinguish two Toxoplasma gondii isolates ("strains" BK and RH). The activity of the following 14 enzymes in the cell lysates was investigated: IDH, MDH, ME, 6PG, G6P, LDH, IPO, HEX, PGM, EST, ALP, ACP, LAP, and PGI. Nine enzymes (IDH, G6P, LDH, HEX, PGM, EST, ALP, ACP, and PGI) showed distinct and reproducible banding patterns, and four of them (IDH, G6P, EST, PGI) enabled a reliable distinction of the two Toxoplasma gondii isolates. A contamination of the parasite extracts with host proteins could be excluded by comparison of the enzyme activities of the Toxoplasma isolates with mouse peritoneal exudate cells. Isoenzyme analysis proved to be a helpful method for a characterization and a distinction of Toxoplasma gondii isolates.

Circulating antigen of Toxoplasma gondii in patients with AIDS: significance of detection and structural properties.

232 sera and 40 cerebrospinal fluid samples of altogether 125 patients in stages III or IV of a HIV-infection were tested for circulating antigen of Toxoplasma gondii by means of a three-layer enzyme-linked immunosorbent assay. Circulating antigen was detected in 32 sera of 20 patients (= 16% of all persons investigated). These ELISA results were reexamined by an Immunoblot following a SDS-PAGE and confirmed in most cases. In addition, this test system led to a partial characterization of the circulating antigen; it consists of at least two proteins with atomic mass units of 27 and 57 kd respectively. The antigenemia was correlated with IgG- and IgM-antibody titres, with clinical symptoms, and with pathological findings also. Our results indicate that the detection of circulating antigen in sera offers a rapid and efficient method for the diagnosis of an acute toxoplasmosis in AIDS-patients.

Comparative studies on the purity and specificity of yolk immunoglobulin Y isolated from eggs laid by hens immunized with Toxoplasma gondii antigen.

Eggs from immunized hens were used as a source of yolk antibodies directed against soluble proteins of Toxoplasma gondii. The yolk immunoglobulins (IgY) were extracted and purified by the use of three different methods. The antibody yield and the purity of each batch were determined by protein measurements, gel filtration and isoelectric focusing. A sequence of two precipitation steps, i.e. a precipitation by polyethylene glycol followed by an alcohol treatment, was shown to be the most effective purification method. The specificity of the yolk antibodies was evaluated by means of an indirect hemagglutination assay, an immunodiffusion test and an immunoelectrophoresis. It was compared with the specificity of IgG antibodies obtained from sera hyperimmunized rabbits. The precipitation patterns of IgY and IgG antibodies were non-identical. This results indicates differences between the specificities of egg yolk IgY antibodies and rabbit IgG serum antibodies, although both animal species had been immunized with identical antigen preparations.

Pneumocystis carinii colonization in the absence of immunosuppression.

A prospective study was undertaken to evaluate the incidence and the course of Pneumocystis carinii colonization in immunocompetent patients with severe pulmonary diseases. A further perspective was to determine the diagnostic values of different detection methods. Bronchoalveolar lavage fluid samples from 77/838 adult HIV-negative patients were examined by Diff-Quik stain, direct immunofluorescence test and polymerase chain reaction. All Diff-Quik stains were negative, but direct immunofluorescence tests and polymerase chain reactions were positive in the samples of 5 patients. The normal number of granulocytes and CD4+T- lymphocytes (median 810 cells/microliters) and normal values of immunoglobulins proved the relative competence of the immune systems of the 77 patients. Although none of these patients received any agent effective against P. carinii, none developed a P. carinii pneumonia within a 120.5-d surveillance period. Nosocomial transmission could be excluded. As the colonization with P. carinii did not result in pneumonia in immunocompetent patients, clinically silent carriers have to be assumed. In non-AIDS patients, sensitive detection methods have to be used to identify colonized persons.

An identical epitope in Pneumocystis carinii and Toxoplasma gondii causing serological cross reactions.

A monoclonal antibody raised against membrane proteins of Toxoplasma gondii with molecular weights of 35 and 21 kDa also reacts strongly and "specifically" with surface antigens of Pneumocystis carinii with molecular weights of 394.2 and 69 kDa when used in a direct immunofluorescence antibody test, on the one hand, and in a immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), on the other. Whether or not this observation might have any phylogenetic relevance remains open.