Anna Lundén

Infectivity of Toxoplasma gondii in mutton following curing, smoking, freezing or microwave cooking

To investigate the effects of curing with sodium chloride and sucrose, low temperature smoking, freezing at -20 degrees C, and cooking in a microwave oven, respectively, on the infectivity of Toxoplasma gondii encysted in mutton, meat from three experimentally and one naturally infected sheep was used. Samples of meat prepared accordingly as well as untreated, raw meat from each animal were assayed by mouse inoculation. Infective T. gondii was isolated from untreated samples from all animals used, but in no case from cured, smoked or frozen meat. However, in two of four steaks processed in a microwave oven, according to a standard household recipe, the parasite remained infective, possibly due to uneven heating of the meat.

Immunisation of mice against neosporosis

In the present study a murine encephalitis model was used to investigate if protection against neosporosis could be achieved by immunisation. Groups of 10 mice were immunised with a sublethal dose of live Neospora caninum tachyzoites, N. caninum antigens incorporated into iscoms, N. caninum lysate mixed with Quil A, or N. caninum lysate in PBS. Control mice were given Quil A only. Challenge infection with 2.5x10(6) N. caninum tachyzoites resulted in clinical symptoms that remained until the end of the experiment in the controls. In contrast, mice immunised with live parasites or parasite lysate in Quil A only showed mild and transient symptoms. Of nine mice immunised with N. caninum iscoms, seven recovered while two died. Most severely affected were the mice immunised with parasite lysate only; all of them died within 28 days post-infection. Histological examination and scoring of brain lesions gave a significantly lower (P<0.0001) lesion score in mice immunised with live parasites than in controls. The groups immunised with iscoms or lysate and Quil A also had reduced lesion scores (P<0.04 and 0.07, respectively) but not the group given parasite lysate alone. The lesions seen in the latter group differed from those in the other groups. There was less cellular reaction and more tachyzoites indicating an active infection. The N. caninum specific antibody responses and cytokine production (IFN-gamma, IL-4 and IL-5) of splenocytes were analysed at the time of challenge infection. The results suggest a correlation between protection and high levels of IFN-gamma. Also, the immune responses recorded in mice immunised with parasite lysate without adjuvant were relatively weak and more towards the Th2 type, when compared with the other immunisation schedules. This is consistent with the weaker inflammatory response observed in the brains of these mice.

Serological Survey of Toxoplasma gondii Infection in Pigs Slaughtered in Sweden

The prevalence of Toxoplasma gondii infection in Swedish pigs was investigated by analysis of 807 meat juice samples collected in 1999 from 10 abattoirs in different parts of the country. When analysed using ELISA, 42 (5.2%) of the samples were found to be positive. The seroprevalence was 3.3% in fattening pigs (n = 695) and 17.3% (n = 110) in adult swine. Alternative interpretations of the results, considering estimates of the true prevalence based on the sensitivity and specificity of the test method, are discussed. It is concluded that the risk of contracting T. gondii infection as a result of eating undercooked pork from Swedish pigs, especially adult animals, is not negligible.

Serological survey of Toxoplasma gondii infection in free-ranging Eurasian Lynx (Lynx lynx) from Sweden

To investigate the prevalence of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) in Sweden, serosanguinous fluids and feces were collected from 207 carcasses of lynx killed or found dead from 1996 to 1998. Sera were tested for antibodies against T. gondii by the direct agglutination test, and 156 (75.4%) of the sera tested positive at antibody titers ≥40. Antibody prevalence was significantly lower in lynx originating from the northern parts of Sweden than in lynx from the more southern regions that are more densely populated by humans. Age-related differences also were found, with a significantly lower prevalence (55%) in juvenile (<1-yr-old) than in subadult and adult animals (82%). There was no significant difference in seroprevalence between males and females. Oocysts typical of T. gondii were not detected in any of the fecal samples.

Immune responses in sheep after immunization with Toxoplasma gondii antigens incorporated into iscoms

An immunization and infection experiment using 12 sheep was conducted to study the immune responses elicited by an experimental vaccine consisting of Toxoplasma gondii antigens incorporated into immunostimulating complexes (iscoms). Five sheep were immunized subcutaneously with Toxoplasma iscoms. Two doses were given, with a 6 week interval, and 22 days after the second immunization, these five sheep and five non-immunized sheep were inoculated orally with T. gondii oocysts. The two remaining animals served as non-immunized, uninfected controls. The antibody response was analysed by an indirect fluorescent antibody test detecting IgM and an enzyme-linked immunosorbent assay detecting IgG. The first immunization induced low levels of both IgM and IgG, and the second resulted in high levels of IgG but no marked IgM response. After infection, a further increase in IgG was observed in the immunized animals. In the non-immunized sheep, substantial IgM and IgG levels were detected following infection. Immunoblotting analysis indicated that the antibody response to immunization was directed against the same T. gondii antigen as the early antibody response after infection in the non-immunized sheep. Antibodies recognizing the P30 antigen appeared first, followed by antibodies to P22 and other antigens which were probably also of membrane origin. Lymphocyte stimulation tests were performed 15 and 21 days after the last immunization and 105 days after infection. Significant antigen-induced proliferative responses were observed after immunization as well as after infection.

Application of iscom antigen preparations in ELISAs for diagnosis of Neospora and Toxoplasma infections

Immunostimulating complexes (iscoms) are cage-like structures of about 40 nm composed of Quil A, cholesterol, phospholipids and antigen. Their main area of use has been as adjuvants and carriers of immunogens in vaccines. Iscoms can also be used for selection of surface membrane proteins of micro-organisms for use in immunoassays, thus decreasing the number of internal proteins that might cause problems with non-specific binding and cross-reactivity. Enzyme-linked immunoassays (ELISAs) utilising parasite antigens incorporated into iscoms have been developed for demonstration of antibodies directed to the intracellular coccidian parasites Toxoplasma gondii and Neospora caninum. These iscom ELISAs have proved very reliable, with high sensitivity and specificity. The preparation of T. gondii and N. caninum iscoms is described, and ELISAs based on iscom antigen preparations that have so far been used for diagnosis of protozoal infections are reviewed.

Serglycin-independent Release of Active Mast Cell Proteases in Response to Toxoplasma gondii Infection

Earlier studies identified serglycin proteoglycan and its heparin chains to be important for storage and activity of mast cell proteases. However, the importance of serglycin for secretion and activity of mast cell proteases in response to parasite infection has been poorly investigated. To address this issue, we studied the effects on mast cell proteases in serglycin-deficient and wild type mice after peritoneal infection with the obligate intracellular parasite Toxoplasma gondii. In line with previous results, we found severely reduced levels of cell-bound mast cell proteases in both noninfected and infected serglycin-deficient mice. However, serglycin-deficient mice secreted mast cell proteases at wild type levels at the site of infection, and enzymatic activities associated with mast cell proteases were equally up-regulated in wild type and serglycin-deficient mice 48 h after infection. In both wild type and serglycin-deficient mice, parasite infection resulted in highly increased extracellular levels of glycosaminoglycans, including hyaluronan and chondroitin sulfate A, suggesting a role of these substances in the general defense mechanism. In contrast, heparan sulfate/heparin was almost undetectable in serglycin-deficient mice, and in wild type mice, it was mainly confined to the cellular fraction and was not increased upon infection. Furthermore, the heparan sulfate/heparin population was less sulfated in serglycin-deficient than in wild type mice indicative for the absence of heparin, which supports that heparin production is dependent on the serglycin core protein. Together, our results suggest that serglycin proteoglycan is dispensable for normal secretion and activity of mast cell proteases in response to peritoneal infection with T. gondii.

Toxoplasma gondii seroprevalence in wild boars ( Sus scrofa ) in Sweden and evaluation of ELISA test performance

SUMMARY Toxoplasma gondii is a zoonotic protozoan parasite, infecting a wide range of warm-blooded animals. The Swedish wild boar population is expanding and increased hunting provides its meat to a growing group of consumers. We performed a spatio-temporal investigation of T. gondii seroprevalence in Swedish wild boars. An ELISA was set up and evaluated against a commercial direct agglutination test, using Bayesian latent class analysis. The ELISA sensitivity and specificity were estimated to 79% and 85%, respectively. Of 1327 serum samples, 50% were positive. Thirty-four per cent of young wild boars and 55% of adults were positive (P < 0·001). The total seroprevalence ranged from 72% in 2005 to 38% in 2011 (P < 0·001), suggesting a declining trend. The highest seroprevalence, 65%, was recorded in South Sweden. In other regions it varied from 29% in Stockholm to 46% in East Middle Sweden.

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Quantitative analysis of parasite DNA in the blood of immunized and naïve mice after infection with Neospora caninum.

Real-time PCR was used to study the duration and level of parasitaemia in mice immunized with immune-stimulating complexes (iscoms) containing recombinant NcSRS2, one of the immunodominant surface antigens of Neospora caninum. After challenge infection, blood was collected daily for 9 days. During this period the amounts of parasite DNA detected in immunized mice were significantly lower (P<0.001), and the duration of parasitaemia appeared to be shorter, than in non-immunized controls. Furthermore, the degree of parasitaemia seemed to correlate well with the amount of N. caninum DNA in the brain 3 weeks post-inoculation and with disease severity measured as changes in body weight. These results indicate that the protective immunity induced by the NcSRS2-iscoms was sufficient to reduce the level of parasitaemia, which probably reduced the number of parasites reaching the brain, and could be the reason for the reduction in brain parasite load and clinical symptoms. Furthermore, real-time PCR was found to be a sensitive means for rapid assessment of N. caninum in blood.