Seroprevalence, Direct Detection and Risk Factors for Toxoplasma gondii Infection in Pigs in Serbia, and Influence of Biosecurity Measures.

Consumption of Toxoplasma gondii contaminated pork is a major risk factor for human infection. We thus conducted a cross-sectional survey on the seroprevalence of T. gondii infection in a representative sample of slaughter pigs from throughout Serbia and examined the influence of farm biosecurity-related risk factors on infection. In addition, direct detection of the parasite (by mouse bioassay) or its DNA was performed in the hearts of a subset of seropositive sows. The overall seroprevalence in the sample of 825 pigs as determined by the modified agglutination test (MAT) was 16.5%. Older age and inadequate rodent control were independent infection risk factors for pigs. In a subset of 581 pigs with complete biosecurity-related data, in addition to older age, smallholders' finishing type farms (as opposed to farrow-to-finish), multispecies farming, and origin from Western and Central and South-Eastern Serbia (vs. the Northern region), all increased the risk of infection, while the absence of disinfection boot-dips in front of each barn and Belgrade district origin (vs. the Northern region) were associated with a 62% and 75% lower risk of infection, respectively. Evidence of viable parasites was obtained in 13 (41.9%) of the 31 bioassayed sow hearts, of which by isolation of brain cysts in seven, by detection of T. gondii DNA in an additional four, and by serology in another two. Recovery of brain cysts mostly (5/7) from sows with a MAT titre of ≥1:100 indicates the risk for consumers. These results highlight the public health risk from pork consumption and point to mandatory use of professional rodent control services, abstaining from multispecies farming, keeping disinfection boot-dips clean and freshly refilled, as well as strict implementation of zoo-hygienic measures on smallholders' farms as specific farm biosecurity measures needed for its reduction.

Experimental infection with Toxoplasma gondii in broiler chickens (Gallus domesticus): seroconversion, tissue cyst distribution, and prophylaxis.

Toxoplasma gondii is a widespread zoonotic protozoan that infects most species of mammals and birds, including poultry. This study aimed to investigate the course of T. gondii infection and the efficacy of diclazuril and Artemisia annua in preventing infection in experimentally infected chickens. Seventy-five 1-month-old chickens, female and male, were randomly divided into five groups (n = 15 each) as follows: (1) uninfected untreated (negative control, NC); (2) infected with T. gondii genotype II/III isolated from a wild cat (group WC); (3) infected with T. gondii genotype II isolated from a domestic cat (group DC); (4) infected with T. gondii domestic cat strain and treated with the anticoccidial diclazuril (group DC-D); and (5) infected with T. gondii domestic cat strain and treated with the medicinal plant Artemisia annua (group DC-A). Clinical signs, body temperature, mortality rate, weight gain, feed conversion ratio, hematological parameters, and the presence of T. gondii-specific IgY antibodies were recorded in all groups. Five chickens per group were euthanized 28 days post-infection (p.i.) and their brains, hearts, and breast muscle tested for T. gondii by mouse bioassay and polymerase chain reaction (PCR). No clinical signs related to the experimental infection were observed throughout the study period. T. gondii-specific antibodies were detected by day 28 p.i., but not in all infected chickens. Overall, T. gondii DNA was detected (bioassay or tissue digests) in all infected and untreated chickens (10/10), while viable parasite (bioassay) was isolated from 7 out of 10 chickens. The parasite was most frequently identified in the brain (7/10). There were no differences in the T. gondii strains regarding clinical infection and the rate of T. gondii detection in tissues. However, higher antibody titers were obtained in chickens infected with T. gondii WC strain (1:192) comparing with T. gondii DC strain (1:48). A. annua reduced replication of the parasite in 3 out of 5 chickens, while diclazuril did not. In conclusion, broiler chickens were resistant to clinical toxoplasmosis, irrespective of the strain (domestic or wild cat strain). The herb A. annua presented prophylactic efficacy by reduced parasite replication. However, further studies are required aiming at the efficacy of diclazuril and A. annua for the prevention of T. gondii infection in chickens using quantitative analysis methods.

Computational image analysis reveals the structural complexity of Toxoplasma gondii tissue cysts.

Toxoplasma gondii is an obligate intracellular parasite infecting up to one third of the human population. The central event in the pathogenesis of toxoplasmosis is the conversion of tachyzoites into encysted bradyzoites. A novel approach to analyze the structure of in vivo-derived tissue cysts may be the increasingly used computational image analysis. The objective of this study was to quantify the geometrical complexity of T. gondii cysts by morphological, particle, and fractal analysis, as well as to determine if it is impacted by parasite strain, cyst age, and host type. A total of 31 images of T. gondii brain cysts of four type-2 strains (Me49, and local isolates BGD1, BGD14, and BGD26) was analyzed using ImageJ software. The parameters of interest included diameter, circularity, packing density (PD), fractal dimension (FD), and lacunarity. Although cyst diameter varied widely, its negative correlation with PD was observed. Circularity was remarkably close to 1, indicating a perfectly round shape of the cysts. PD and FD did not vary among cysts of different strains, age, and derived from mice of different genetic background. Conversely, lacunarity, which is a measure of heterogeneity, was significantly lower for BGD1 strain vs. all other strains, and higher for Me49 vs. BGD14 and BGD26, but did not differ among Me49 cysts of different age, or those derived from genetically different mice. The results indicate a highly uniform structure and occupancy of the different T. gondii tissue cysts. This study furthers the use of image analysis in describing the structural complexity of T. gondii cyst morphology, and presents the first application of fractal analysis for this purpose. The presented results show that use of a freely available software is a cost-effective approach to advance automated image scoring for T. gondii cysts.

Source attribution of human echinococcosis: A systematic review and meta-analysis.

BACKGROUND: A substantial proportion of echinococcosis transmission to humans via contamination of food has been assumed. However, the relative importance of food as a transmission vehicle has previously been estimated through expert opinion rather than empirical data. OBJECTIVE: To find and evaluate empirical data that could be used to estimate the source attribution of echinococcosis, in particular the proportion that is transmitted through contaminated food. METHODS: A systematic review was undertaken to identify reports on the risk factors for human cystic (CE) and alveolar (AE) echinococcosis. Data bases searched included PubMed, Scopus, Web of Knowledge, Cab Direct, Science Direct, Google Scholar, eLIBRARY.RU, CyberLeninka, CNKI and VIP. Search terms included Echinococc*, hydatid, epidemiology, logistic regression, risk factors, odds ratio, relative risk, risk factors. Reports, including grey literature where available, that had suitable data were selected and data were extracted. The main pathways of transmission were hypothesised to be contact with the definitive host, contaminated water, contaminated food and contaminated environment (other than food). For each study the attributable fraction for these potential sources of infection was calculated from the data presented. A meta-analysis was then undertaken to obtain pooled estimates for the relative contribution of these transmission pathways. RESULTS: Data from 28 cross-sectional studies and 14 case-control studies were extracted. There was strong evidence for transmission by direct contact with dogs for both CE and AE. The estimated attributable fractions were 26.1% (CI 13.8%-39.6%) and 34.4% (CI 20.7% -48.2%) respectively. Transmission through contaminated water was estimated to be responsible for approximately 29.4% (CI 12.1%-51.7%) for CE and 24.8% (CI 10.6% to 42.6%) for AE. Contaminated food may be responsible for approximately 23.4% of CE cases (CI 2.1%-47.3%). Globally, there was insufficient evidence to conclude AE can be transmitted by food, although case control studies from low human incidence areas suggested that possibly 32.5% (CI 10.0%-53.2%) could be transmitted by food. There was also insufficient evidence that direct contact with foxes was a significant source of human disease. There were no suitable studies with a risk of environmental contact reported, but the residual attributable fraction thatwould likely include this pathway was approximately 21.1% for CE and 11.1% for AE. CONCLUSIONS: The results support the hypothesis that dog contact and drinking contaminated water are major pathways of transmission of both CE and AE. For contaminated food, the results are less consistent, but suggest that it is an important transmission pathway and provide better evidence than expert elicitations as previously used.

Taeniosis and cysticercosis in Serbia, 1990-2018: Significance of standard of living.

OBJECTIVES: As is the case for all of Southeast Europe, Serbia is an area traditionally endemic for Taenia saginata and Taenia solium infections. This study was performed to analyse the epidemiological data on taeniosis and cysticercosis in Serbia for the period 1990-2018. METHODS: Data on cases of T. saginata and T. solium infection were collected via a systematic search of published articles, the grey literature, and official reports, as well as by performing clinical observational studies of patients treated in the departments for infectious diseases of hospitals and university clinics in Serbia. RESULTS: A total of 212 cases of taeniosis were reported, all between 1997 and 2004 when taeniosis was notifiable (incidence range 0.04-0.9/100 000 population/year). From 1990 to 2018, 170 cases of cysticercosis (all but one of neurocysticercosis), were registered (incidence range 0-0.29/100 000 population/year), with a strong decrease since 2000 and a single case in the last 9 years. The annual number of cases of both taeniosis (Pearson's r = 0.914, p = 0.001) and cysticercosis (Pearson's r = 0.582, p = 0.014) correlated with the consumer price index. CONCLUSIONS: In Serbia, T. saginata and T. solium infections are autochthonous but occur only sporadically. However, the potential for re-emergence exists, depending on the socio-economic state of the country.

Detection of Toxoplasma gondii in naturally infected domestic pigs in Northern Serbia.

Insufficiently cooked pork is considered as an important source of human infection with Toxoplasma gondii. The aim of our study was to investigate the presence of T. gondii in pigs intended for human consumption from Northern Serbia. Blood and diaphragm samples were collected from 182 naturally infected market-weight pigs, originating from both commercial farms and smallholdings. Sera were examined using modified agglutination test (MAT), and diaphragms from seropositive, as well as from some MAT-negative pigs, were bioassayed in mice. In addition, digests were examined for the presence of T. gondii DNA using a real-time polymerase chain reaction (qPCR) which was targeted at the 529 bp repetitive element of the T. gondii genome. The overall seroprevalence of T. gondii in pigs was 17% (31/182), with no difference between pigs from large commercial farms (17.8%) and those raised on smallholdings (16.3%). However, the seroprevalence in farm pigs was largely influenced by the findings on a single farm, where all examined animals tested positive. Parasites and/or parasite DNA were detected in the tissues of 15 of the 45 (25 seropositive and 20 seronegative) animals examined by either direct method. Tissue cysts were isolated in eight bioassays and an additional bioassay was positive by serology; all nine were confirmed positive by qPCR. All positive bioassays originated from seropositive pigs, but no correlation was observed between isolation rate and antibody titer. T. gondii DNA was detected in diaphragm tissues of eight pigs, of which three were seronegative. The results of our study provide further evidence for pork as a source of human T. gondii infection.

The first isolation and molecular characterization of Toxoplasma gondii from horses in Serbia.

BACKGROUND: Consumption of undercooked or insufficiently cured meat is a major risk factor for human infection with Toxoplasma gondii. Although horsemeat is typically consumed rare or undercooked, information on the risk of T. gondii from infected horse meat to humans is scarce. Here, we present the results of a study to determine the presence of T. gondii infection in slaughter horses in Serbia, and to attempt to isolate viable parasites. METHODS: The study included horses from all regions of Serbia slaughtered at two abattoirs between June 2013 and June 2015. Blood sera were tested for the presence of specific IgG T. gondii antibodies by the modified agglutination test (MAT), and samples of trypsin-digested heart tissue were bioassayed in mice. Cyst-positive mouse brain homogenates were subjected to DNA extraction and T. gondii strains were genotyped using 15 microsatellite markers (MS). RESULTS: A total of 105 slaughter horses were sampled. At the 1:6 cut-off 48.6% of the examined horses were seropositive, with the highest titre being 1:400. Viable parasites were isolated from two grade type mares; both parasite isolates (RS-Eq39 and RS-Eq40) were T. gondii type III, and both displayed an increased lethality for mice with successive passages. These are the first cases of isolation of T. gondii from horses in Serbia. When compared with a worldwide collection of 61 type III and type III-like strains, isolate RS-Eq39 showed a combination of MS lengths similar to a strain isolated from a duck in Iran, and isolate RS-Eq40 was identical in all markers to three strains isolated from a goat from Gabon, a sheep from France and a pig from Portugal. Interestingly, the source horses were one seronegative and one weakly seropositive. CONCLUSIONS: The isolation of viable T. gondii parasites from slaughter horses points to horsemeat as a potential source of human infection, but the fact that viable parasites were isolated from horses with only a serological trace of T. gondii infection presents further evidence that serology may not be adequate to assess the risk of toxoplasmosis from horsemeat consumption. Presence of T. gondii type III in Serbia sheds more light into the potential origin of this archetypal lineage in Europe.

Prenatal and Early Postnatal Diagnosis of Congenital Toxoplasmosis in a Setting With No Systematic Screening in Pregnancy.

To determine the risk of congenital toxoplasmosis (CT) and provide early (pre- or postnatal) identification of cases of CT in the absence of systematic screening in pregnancy.I n the presented cross-sectional study, serological criteria were used to date Toxoplasma gondii infection versus conception in 80 pregnant women with fetal abnormalities or referred to as suspected of acute infection, and in 16 women after delivery of symptomatic neonates. A combination of serological, molecular (qPCR), and biological (bioassay) methods was used for prenatal and/or postnatal diagnosis of CT. Most (77.5%) pregnant women were examined in advanced pregnancy. Of all the examined seropositive women (n = 90), infection could not be ruled out to have occurred during pregnancy in 93.3%, of which the majority (69%) was dated to the periconceptual period. CT was diagnosed in 25 cases, of which 17 prenatally and 8 postnatally. Molecular diagnosis proved superior, but the diagnosis of CT based on bioassay in 7 instances and by Western blot in 2 neonates shows that other methods remain indispensable. In the absence of systematic screening in pregnancy, maternal infection is often diagnosed late, or even only when fetal/neonatal infection is suspected. In such situations, use of a complex algorithm involving a combination of serological, biological, and molecular methods allows for prenatal and/or early postnatal diagnosis of CT, but lacks the preventive capacity provided by early maternal treatment.

Echinococcosis in Serbia: an issue for the 21st century?

Echinococcosis (hydatidosis) is traditionally endemic in Southeast Europe, Serbia included. In Serbia, echinococcosis is mandatory reportable, and this review analyzes the officially reported data as well as the research data published between 1998 and 2010. Official data on human and animal infections were obtained from the Institute of Public Health of Serbia (IPHS, 2010), and from the Ministry of Agriculture, Trade, Forestry and Water Management (MATFWM) and the Statistical Office, respectively. Published data were obtained by searching the Medline, Scopus, and Google databases using "echinococcosis," "hydatidosis," and "Serbia" as key words. In addition, the search included national journals and doctoral theses, as well as conference proceedings. Only Echinococcus granulosus has been reported in Serbia, with a total of 409 cases of human infection officially reported during the observed period as opposed to 820 cases described in clinical studies. No trend in the incidence of infection was shown among adults, but the number of cases in children continuously decreased over the period. Patients were more frequently female and from rural areas. Differences in the geographic distribution of cases were noted, with a lower incidence in the central part of country. Liver disease was by far the most common presentation, but cases of unusual cyst locations have been described. Among domestic animals, sheep were the most highly infected species. A decreasing incidence of echinococcosis in animals has been noted as of the 1970s. Echinococcosis continues to be endemic in Serbia in the 21st century, but despite predictions, neither official data nor those from clinical studies indicate its re-emergence. However, there is gross underreporting. Public health authorities should actively work to increase reporting, as only valid reported data provide an accurate basis for future control plans.

Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis.

A sensitive real-time PCR technique was used to examine the distribution of Toxoplasma gondii in the blood and tissues of mice during acute and chronic infection. Groups of Swiss Albino mice, inoculated i.p. with 10(2) or 10(6) tachyzoites of the RH strain as a typical type-1 strain, or fed 10 cysts of the Me49 strain as a typical type-2 strain, were killed at different time points post-infection (p.i.), and blood and organs including the lungs, brain and liver were harvested for DNA extraction. Toxoplasma DNA was quantified by a real-time PCR targeted at the 529bp gene fragment, with a detection limit of a single parasite per g/ml of tissue. The results showed a strain- and dose-dependent spread of Toxoplasma. In infection with type-1 parasites, in case of a high infective dose, Toxoplasma DNA was detected within 24h p.i. in all analyzed tissues including the brain. Conversely, in case of a low infective dose, parasitaemia was undetectable early p.i., at a time when Toxoplasma DNA was detected in the tissues, but reached very high levels as infection progressed. With both infective doses, pre-death parasite burdens were higher in the blood than in the tissues, whereas the same loads in the lungs suggest that reaching these Toxoplasma burdens may be critical for survival. In infection with Me49 parasites, steady high parasite burdens were noted up to the end of the experiment at d42 only in the brain, parasitaemia was low but detectable throughout, and Toxoplasma DNA was completely cleared only from the liver. These data are important to better understand the pathogenesis of toxoplasmosis, and also as baseline data for the experimental evaluation of novel chemotherapeutics.

Adverse fetal outcome in the absence of timely prenatal diagnosis of congenital toxoplasmosis.

OBJECTIVE: Toxoplasma gondii infection acquired during pregnancy may lead to transplacental transmission and jeopardize the course and outcome of pregnancy, leading to life-threatening disease in the fetus and the newborn. CASE REPORT: Here we present a case of medically terminated pregnancy due to clinically manifested congenital toxoplasmosis (CT) which was proven serologically, as well as by bioassay. Ultrasonographically visualized severe fetal ventriculomegaly in a seven-month pregnant 33-year-old woman with a history of three months of lymphadenopathy was an indication for extensive testing for toxoplasmosis. Based on the serological results obtained (high specific IgG antibodies of borderline but close-to-low avidity, along with the finding of specific IgM antibodies), maternal infection was dated to the second trimester. Cord blood serology revealed IgG levels lower than those of the mother's, but both specific IgM and IgA antibodies were detected, indicating fetal infection. Although Toxoplasma DNA was not detected in the cord blood sample by real-time PCR, fetal infection was definitely confirmed after six weeks by cord blood bioassay results. While no morphologically recognizable Toxoplasma cysts were found, murine serology was positive. Since fetal morphological abnormalities, which could not be reversed by subsequent treatment, were already advanced at the time of serological testing, the patient opted for termination of pregnancy. CONCLUSION: This case demonstrates the potentially severe outcome of CT as a result of central nervous system affection, emphasizing the need for prompt and precise prenatal diagnosis in case of maternal seroconversion, so that proper treatment may be introduced in a timely manner.

Toxoplasma gondii infection in slaughter pigs in Serbia: seroprevalence and demonstration of parasites in blood.

A seroepizootiological study of Toxoplasma gondii infection involving a total of 488 slaughter pigs (468 market-weight pigs and 20 sows) in the Belgrade area, also included examination of the presence of T. gondii in the blood. Blood sampled at the slaughter line was examined for specific antibodies by modified direct agglutination, and blood clots of those seropositive at titres of 1:50-1:12800 were bioassayed in mice. The overall seroprevalence was 9.2%, significantly higher (p = 0.0063) in sows (30.0%) than in market-weight pigs (8.3%). Amongst the 22 bioassays performed, a total of 16 (72.7%) were positive, by observation of T. gondii cysts (12), seropositivity (7, including 3 in which cysts were not detected), and/or detection of T. gondii DNA by real-time PCR (12, including one otherwise negative). The positive bioassays originated from the blood of 12 market-weight pigs and 4 sows. Despite a general increase in the rate of demonstration of T. gondii with the increase in the specific antibody level, the association was not significant (p = 0.101). The risk of infection was 41-fold increased in sows vs market-weight pigs, and 15-fold in pigs from smallholders' finishing type farms vs those from large farrow-to-finish farms. The presence of viable T. gondii in a proportion of the samples indicates that some of the pigs had an active parasitaemia at the time of slaughter, which, along with the seroprevalence established, points to a potential source of human infection in Serbia. This is the first report on parasitaemia in naturally infected swine.

Toxoplasmosis in naturally infected rodents in Belgrade, Serbia.

To assess the role of synanthropic rodents in the epidemiology of urban toxoplasmosis, Toxoplasma gondii infection was examined in 144 rats (Rattus norvegicus) and 12 mice (Mus musculus) captured using live animal traps in three locations in Belgrade city characterized by poor housing and degraded environment. In rats, specific IgG antibodies were detected by modified agglutination test in 22 (27.5%) of the 80 blood samples available. Toxoplasma brain cysts were microscopically detected in 11 (7.6%), and Toxoplasma DNA by real-time polymerase chain reaction was demonstrated in 15 (10.4%) animals. Of these, both cysts and Toxoplasma DNA were detected in five (3.5%) rats. In mice, cysts were observed in 3 (25%), but Toxoplasma DNA was detected in even 10 (83.3%) animals, including all 3 with morphologically recognized cysts. Being a link in the chain of Toxoplasma infection, the existence of urban rodent reservoirs of infection represents a public health risk.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host.

Host lipids have been implicated in the pathogenesis of Toxoplasma gondii infection. To determine if Toxoplasma infection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 micromol/L, with a mean of 2.43 +/- 4.14 micromol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44 percent mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

Comparative evaluation of three commercial Toxoplasma-specific IgG antibody avidity tests and significance in different clinical settings.

Determination of the avidity of specific IgG antibodies has become a generally accepted diagnostic aid for dating Toxoplasma infection. In this study, the Labsystems, VIDAS and EUROIMMUN Toxoplasma IgG avidity assays were compared on a series of 133 Toxoplasma IgG- and IgM-positive sera from symptomatic patients (n=28), from pregnant (n=43) and non-pregnant (n=26) women, and on 18 IgG-positive and IgM-negative sera from chronically infected patients. The results showed excellent concordance between the Labsystems and VIDAS tests in both the IgM-positive (r=0.82, kappa=0.771) and IgM-negative (kappa=0.609) sera, whilst the agreement of the EUROIMMUN assay with both the Labsystems and VIDAS tests in the IgM-positive sera was moderate (kappa=0.575 and kappa=0.525, respectively) and in the IgM-negative sera was poor (kappa=0.000). Analysis of the kinetics of the maturation of avidity in 13 patients in whom follow-up sera were available showed that, despite a general trend of maturation, in two patients the avidity did not become high during 6 and 11 months of follow-up. In view of the clinical setting, in the symptomatic patients, despite one case of complete discrepancy and five cases of partial discrepancy, the Labsystems and VIDAS tests were in almost perfect agreement (kappa=0.812), whilst the agreement in pregnant and non-pregnant women was substantial (kappa=0.754 and kappa=0.708, respectively). In conclusion, the Labsystems and VIDAS tests are equally reliable for the measurement of Toxoplasma IgG avidity; the choice of test should depend on the laboratory set-up. The EUROIMMUN test may be an acceptable alternative in resource-limited settings, but should be used prudently.

A human origin type II strain of Toxoplasma gondii causing severe encephalitis in mice.

Despite its capacity for sexual reproduction and global distribution, Toxoplasma gondii has a highly clonal structure, with the majority of isolates belonging to three distinct clonal types. Congenital toxoplasmosis has been associated with type I and type II strains. We here present the first characterization of a T. gondii strain (BGD1) from South-East Europe, isolated from the umbilical blood of a 24-week-old fetus in Serbia. Genotyping, performed by PCR-RFLP using a set of nested PCR markers (5'SAG2, 3'SAG2, BTUB, SAG3, and GRA6), showed that the BGD1 strain possessed a type II genotype. The cytokine patterns in Swiss-Webster mice inoculated with brain cysts of BGD1 and the prototype type II ME49 strain were similar until 180 days post-infection, with highly elevated IFN-gamma, IL-12 and IL-10 by d7 and decreasing thereafter. While both strains induced pneumonia and hepatitis in acute infection (d14), chronic infection (d56) was characterized, in addition to hepatitis, by severe meningoencephalitis, associated with numerous brain cysts. Thus, the BGD1 strain of T. gondii has type II genotypic and immunologic characteristics, but unlike other type II strains of human origin, induces severe encephalitis, making it an alternative to the sheep-derived ME49 strain for experimental models of infection.

Effectiveness of spiramycin in murine models of acute and chronic toxoplasmosis.

The antitoxoplasmic activity of spiramycin (SPI) was evaluated in murine models of infection using a type-1 (RH) or type-2 (Me49) strain of Toxoplasma gondii. In mice infected with 10(2) tachyzoites of the RH strain, treatment with 100 and 200 mg SPI/kg/day had only a limited effect; despite some dose-dependent prolongation of survival, it was unable to protect mice against death. In contrast, in acute infection induced by peroral inoculation of 10, but not 20, cysts of the Me49 strain, a 3-week course of 100 mg SPI/kg/day and a 4-week course of 200 mg/kg/day significantly enhanced protection and markedly reduced brain cyst burdens at 6 months post infection (p.i.). In chronic infection established by inoculation of 10 cysts 3 months previously, a 3-week course of 200 mg SPI/kg/day resulted in significantly decreased brain cyst burdens compared with controls, both 2 weeks after treatment cessation and by 6 months p.i. Although a favourable effect on chronic infection may be specific for mice, these data merit investigation, since they may have clinical ramifications.

Stage conversion of Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine dithiocarbamate.

The mouse-virulent RH strain of Toxoplasma gondii is generally considered to have lost its cyst-forming capacity, and conversion of RH tachyzoites into cysts in non-immune mice has previously been shown exclusively following early treatment with sulfadiazine (SDZ). We here describe the development of tissue cysts in mice infected with RH strain parasites and treated with atovaquone (ATO) combined with pyrrolidine dithiocarbamate (PDTC). Groups of Swiss-Webster mice infected intraperitoneally (i.p.) with 10(2) RH tachyzoites were treated with 5, 25 and 100 mg of ATO/kg per day alone or combined with PDTC at 250 mg/kg per day from day 1 postinfection (p.i.) for 14 days. A total of 19 mice survived the 6-week observation period. Of these, brain cysts were recovered in nine (47%), with burdens ranging from 50 to 3120 (mean +/- S.D. = 622 +/- 963). All cyst-harboring mice had high specific IgG antibody levels (1:10,240-1:40,960, corresponding to 500-2000 IU/ml), as did one mouse in which cysts were not demonstrated, which was therefore included in the group of mice with residual infection. Bioassay performed to test the infectivity of these cysts produced acute lethal toxoplasmosis following i.p. inoculation in all instances (100%), and importantly, following peroral inoculation in four (29%). The recovered tachyzoites were highly infectious. In addition, significantly elevated interferon gamma (IFN-gamma) in the treated mice which developed residual infection compared with any group of infection-free (treated or subinoculated) mice, indicates immunological control of the parasite in the latent form. In conclusion, early treatment of mice infected with T. gondii RH tachyzoites with ATO and PDTC induces conversion into tissue cysts, thus providing a new model for studying the mechanism(s) of T. gondii stage conversion.