Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Monitoring of local CD8ß-expressing cell populations during Eimeria tenella infection of naïve and immune chickens.

The purpose of this study was to monitor abundance and activation of local CD8ß-expressing T-cell populations during Eimeria tenella infections of naïve chickens and chickens immune by previous infections. Chickens were infected with E. tenella up to three times. Caecal T-cell receptor (TCR) γ/δ-CD8ß+ cells (cytotoxic T lymphocytes; CTL) and TCRγ/δ+CD8ß+ cells were characterized with respect to activation markers (blast transformation, CD25 and cell surface CD107a). Cells were also induced to degranulate in vitro as a measure of activation potential. Major findings included a prominent long-lasting, up to 6 weeks, increase in the proportion of CTL among caecal CD45+ cells in the later stages after primary E. tenella infection. These CTL also showed clear signs of activation, that is blast transformation and increased in vitro induced degranulation. At second and third E. tenella infection, chickens showed strong protective immunity but discrete signs of cellular activation were observed, for example increased in vitro induced degranulation of CTL. Thus, primary E. tenella infection induced clear recruitment and activation of local CTL. Upon subsequent infections of strongly immune chickens cellular changes were less prominent, possibly due to lower overall numbers of cells being activated because of the severe restriction of parasite replication.

Expression of perforin, granzyme A and Fas ligand mRNA in caecal tissues upon Eimeria tenella infection of naïve and immune chickens.

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella-infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which were manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection, perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast, at infection of immune birds, perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1-4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T cells in the protection against Eimeria infections.

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

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.

Cytokine mRNA expression in bronchoalveolar lavage cells during Dictyocaulus viviparus infection in calves.

The purpose of this study was to monitor local cytokine responses to Dictyocaulus viviparus in calves during primary infection and re-infection. Bronchoalveolar lavage fluid (BALF) was collected weekly from experimentally infected calves and interleukin-2 (IL-2), IL-4, IL-5, IL-10 and IFN-γ mRNA expression was quantified in BALF cells. The major finding was a prominent transient increase in IL-4 mRNA expression, compared with that of uninfected calves, observed in BALF cells collected 2-3 weeks post-primary D. viviparus infection. At 2 weeks post-infection, macroscopic worms were also first observed in BALF. Calves re-infected after 10 weeks were partially immune which was evident at slaughter 5 weeks post-infection as a lower worm burden than in previously naïve calves infected at the same time. IL-4 mRNA expression in BALF cells 2 weeks post-re-infection was increased compared with that of uninfected animals but not as high as that of primarily infected calves. BALF cell expression of the other cytokines tested for was not as clearly effected by the D. viviparus infection. It seems likely that the strong IL-4 response observed during primary infection reflects an innate response to the worms that may initiate an ensuing Th2 response, which confers protective immunity.

Association of DGAT1 genotype, fatty acid composition, and concentration of copper in milk with spontaneous oxidized flavor.

In 136 cows with altogether 969 milk samples, we investigated the effect of the acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism on milk fatty acid (FA) composition and how, in combination with copper concentration in milk, this influences the occurrence of spontaneous oxidized flavor. All milk samples were analyzed for concentrations of copper and individual FA and subjected to sensory analysis by trained judges. We found an effect of DGAT1 genotype on FA composition where mainly the long-chain FA were affected. The 232A allele was associated with larger proportions of the C18:2 cis-9,trans-11 conjugated linoleic acid and lower proportions of C16:0 FA. Milk concentrations of unsaturated FA and copper showed strong and unfavorable associations with spontaneous oxidized flavor (SOF) development. The interaction between FA and copper indicates that SOF will not develop as easily in milk with high copper content unless the substrate is available (i.e., in addition to the previously shown effect of copper in milk, unsaturated FA are required for the process of oxidation to progress). We observed a marked effect of the DGAT1 genotype on SOF development where the A allele was associated with a higher risk of SOF. Moreover, our results suggest that the effects of the FA C18:3 n-3 and of the polyunsaturated index on SOF development are beyond the effect of the DGAT1 genotype. Breed had an effect on FA composition but not on SOF development. Our results imply that copper, FA composition, and DGAT1 genotype are risk factors for SOF and considerations to these factors might be necessary in future breeding decisions.

Genetic analyses of pathogen-specific mastitis.

The aims of this study were to investigate the presence of genetic variation for susceptibility to pathogen-specific mastitis and to examine whether haplotypes of an identified quantitative trait locus with effect on unspecific mastitis resistance had different effects on specific mastitis pathogens. Bacteriological data on mastitis pathogens were obtained from the diagnostic laboratory at the Swedish National Veterinary Institute. The data were mainly from subclinical cases of mastitis but also clinical cases were included. Variance components were estimated for incidence of the six most frequent pathogens using Markov Chain Monte Carlo methodology via Gibbs sampling. Genetic variation for susceptibility to pathogen-specific mastitis was higher compared to estimates of general resistance to clinical mastitis in most other studies. However, because of the non-random nature of data collection, comparisons to other studies should be made by caution. The effect of haplotype on the risk of being infected by a given mastitis pathogen, relative to other pathogens, was studied using an allele substitution model. Although there were no significant haplotype substitution effects on the resistance to any of the six mastitis pathogens, there was a significant difference between the effects of two of the haplotypes regarding the risk of acquiring a Streptococcus dysgalactiae infection.

Molecular classification and biomarkers of clinical outcome in breast ductal carcinoma in situ: Analysis of TBCRC 038 and RAHBT cohorts.

Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We perform multiscale, integrated molecular profiling of DCIS with clinical outcomes by analyzing 774 DCIS samples from 542 patients with 7.3 years median follow-up from the Translational Breast Cancer Research Consortium 038 study and the Resource of Archival Breast Tissue cohorts. We identify 812 genes associated with ipsilateral recurrence within 5 years from treatment and develop a classifier that predicts DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions are identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

Characterization of bovine lymphocytes stimulated in vitro by Dictyocaulus viviparus homogenate.

Adult Dictyocaulus viviparus homogenate induced proliferation of lymphocytes from naïve cattle. We characterized the responding cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) loading, for detection of proliferation, and antibody labelling for cell surface molecules. Lymphocytes expressing CD4, CD8 and gamma/delta TCR, rather than Ig expressing cells, proliferated after in vitro stimulation with D. viviparus homogenate. Of gamma/delta TCR expressing cells, both CD8, WC1.1 and WC1.2 co-expressing cells proliferated. Moreover, gamma/delta T cells expressing MHC class II proliferated to a higher extent than those negative for MHC class II. Of CD4 and CD8 expressing lymphocytes, both those co-expressing CD45R and CD45R0 proliferated. Among CD4 expressing lymphocytes, those that were CD45R0 positive had a larger proportion of proliferated cells than did CD45R positive cells. Compared to stimulation with Con A, the proportion of dividing cells after D. viviparus stimulation was smaller although the cells had divided more times. Furthermore, we also compared in vitro responses of peripheral blood mononuclear cells collected before and after two subsequent infections with D. viviparus, but no clear acquired responses could be detected. Overall, this suggests that most T lymphocytes are stimulated by the D. viviparus homogenate rather than any particular lymphocyte subpopulation.

Frequency and effect of the bovine acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism in Swedish dairy cattle.

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol synthesis in the mammary gland, and the corresponding gene has emerged as a strong candidate for the variation in milk fat percentage. In this study, the allele frequencies and effects of the DGAT1 K232A variants in the Swedish dairy breeds Swedish Red and Swedish Holstein were investigated. A total of 239 cows, 143 of the Swedish Red breed and 96 of the Swedish Holstein breed, in the experimental herd at the Swedish University of Agricultural Sciences were genotyped for the DGAT1 polymorphism. The Swedish Red cows in the herd belonged to 1 of 2 selection lines with high or low milk fat percentage, respectively, but with similar high total milk energy production. The frequency of the K variant was found to be significantly greater in the high-fat line than in the low-fat line. The average frequency of the K variant in the 2 lines of the Swedish Red cows was 0.09 compared with 0.12 among the Swedish Holstein cows. Mixed model analysis was used to estimate the effect of the DGAT1 K232A polymorphism based on 16,866 test-day records for milk production traits. In accordance with previous studies, the most pronounced effects were found for fat and protein percentages and milk yield; and the K variant was associated with an increase in milk fat and protein percentages but less milk yield compared with the A variant. Less pronounced effects were found for yields of fat and protein for which the K variant was associated with greater fat yield but less protein yield.

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.

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.

Methylsulfonyl metabolites of PCBs and DDE in human milk in Sweden, 1972-1992.

A multicomponent method used for analysis of organochlorine pesticides, polychlorinated biphenyls (PCBs), naphthalenes, dibenzo-p-dioxins, and dibenzofurans was adapted for the analysis of methylsulfonyl metabolites of chlorinated biphenyls (MeSO2-CBs) and of p,p'-DDE (MeSO2-DDE) in human milk. The extraction and initial purification was made by liquid-gel partitioning. Additional purification and separation steps were achieved by adsorption and gel permeation chromatography. The mean recoveries of 23 MeSO2-CBs and MeSO2-DDE standards, added to the milk before extraction, were 80-97%. Human milk sampled in Stockholm during 1972, 1976, 1980, 1984/85, 1990, 1991, and 1992 was analyzed by GC-MS. During the time course studied, the concentrations of MeSO2-CBs decreased from approximately 9 to 2 ng/g lipids and of MeSO2-DDE from 5 to 0.4 ng/g lipids. The concentrations of MeSO2-CBs and MeSO2-DDE correlated to the levels of total PCB and p,p'-DDE, respectively. 3-MeSO2-DDE was the major isomer of the aryl methyl sulfones studied in the milk. PCB methyl sulfones with five and six chlorine atoms in the molecule were predominant among the PCB methyl sulfones Generally, the concentrations of 4-MeSO2-CBs were higher than the corresponding 3-MeSO2-CB compound. The major MeSO2-CBs in the milk were 4-MeSO2-2,5,2',3',4'-pentaCB (4-87) and 4-MeSO2-2,3,6,2',4',5'-hexaCB (4-149).

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.

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.

Experimental Toxoplasma gondii infection leading to fatal enteritis in reindeer (Rangifer tarandus).

Two, 50-60-kg yearling reindeer (Rangifer tarandus) were inoculated intraruminally with 5,000 (reindeer 1) or 50,000 (reindeer 2) oocysts of the ME-49 strain of Toxoplasma gondii. Both reindeer became ill day 4 postinoculation (p.i.). Reindeer 2 died because of acute enteritis day 9 p.i. Histologically, extensive necrosis and destruction of the intestinal mucosa were seen. Numerous T. gondii organisms were demonstrated immunohistochemically. Reindeer 1 was treated with sulfatrimethoprim for 2 days from day 9, recovered by day 16 p.i., and remained clinically normal until the last day of observation (day 707 p.i.). It developed high antibody titers to T. gondii between days 7 and 14 p.i.

Associations between major histocompatibility complex genes and production traits in White Leghorns.

The frequency of the MHC haplotype B15 had been found in a previous study to be more than two times higher in a White Leghorn line selected for high egg production compared with the unselected control strain. To further evaluate these findings, matings were performed between chickens with the same heterozygous B genotypes, being combinations of the most frequent haplotypes, i.e., B15, B19, and B21. In total, more than 1,300 observations from two generations were analyzed. In each generation, approximately one half of the chickens were derived from the line selected for total egg mass, the other half from the control strain. The MHC genotypes were determined serologically. Additive and dominance effects of B haplotypes on production traits were analyzed using an individual animal model. The estimation of genotypic values, together with the analysis of gene substitution effects, showed that the B15 haplotype was associated with early sexual maturity and low egg production during the late production period, i.e., between 43 and 63 wk of age, whereas B19 was associated with later onset of sexual maturity. The association of B15 with early sexual maturity would thus explain the high frequency of the B15 haplotype previously observed in a line selected for high early egg production. No dominance effect of the B system was observed for any of the traits, suggesting that the present results were due predominantly to additive gene effects.

Sporulation of Eimeria maxima oocysts in litter with different moisture contents.

The aim of this study was to determine if the sporulation of Eimeria maxima oocysts was affected by the moisture content of the litter. Fresh feces were collected from chickens experimentally infected with E. maxima. The feces were mixed with dried wood shavings and different amounts of water to obtain final moisture contents of 16, 42, and 62% and a final oocyst concentration of 5,000 per g of mixture. The samples were kept at 23 C and 75% relative humidity and were thoroughly aerated every 12 h. Oocysts kept under ordinary laboratory sporulation conditions in 2% wt/vol aqueous potassium dichromate at 27 C were used as a standard for optimal sporulation. The proportion of sporulated oocysts was determined microscopically every 12 h. Sporulation of E. maxima was most efficient under the driest conditions studied (16% moisture content), and poorest in the samples with the highest moisture content (62%). Even though the differences may not have resulted from a direct effect of humidity on the oocysts, but more likely resulted from limited oxygen in the moister substrates, it is clear that sporulation is not favored by moist litter.

Long-term study of Toxoplasma gondii infection in a Swedish sheep flock.

The infection rate of Toxoplasma gondii was studied during 6 years in a sheep flock in central Sweden. The flock consisted of 165-249 breeding ewes of which 20-35% were lambs less than 1 year old. Most ewes were slaughtered when 5 years old. The sheep were kept indoors from end of September to early May. Lambing took place in March and April. Individual serum samples were collected twice a year, once just before turning the sheep out to pasture in the spring and again after housing in the autumn. Sera were analysed by ELISA for antibodies to T. gondii. The seroprevalence varied between 10% and 45% during the 6 years of observation. Seroconversion was detected predominantly at the autumn sampling, indicating that in most cases infection was acquired at pasture. Subclinical effects of T. gondii infection on lamb weight, litter size, total litter weight and ewe weight were also studied. Lambs born to chronically infected ewes were lighter at birth than those of uninfected ewes, but this disparity was no longer evident at weaning.