Chicken line-dependent mortality after experimental infection with three type IIxIII recombinant Toxoplasma gondii clones.

Three genetically different clones of Toxoplasma gondii, also different in mouse virulence, were studied by experimental infection in chickens. For the experiments, four chicken lines were used, which differed in phylogenetic origin and performance level: two white egg layer lines, one with high laying performance (WLA), one with low (R11) and two brown layer lines, also displaying high (BLA) and low (L68) egg number. Chickens were intraperitoneally infected with three different T. gondii isolates representing type IIxIII recombinant clones, i.e. showing both, type II- and type III-specific alleles. These clones (K119/2 2C10, B136/1 B6H6, K119/2 A7) had exhibited virulence differences in a mouse model. In chickens, a significantly higher mortality was observed in white layer lines, but not in brown layer lines, suggesting that differences in the phylogenetic background may influence the susceptibility of chickens for toxoplasmosis. In addition, antibody (IgY) levels varied in surviving chickens at 31 days post infection. While low to intermediate antibody levels were observed in white layers, intermediate to high levels were measured in brown layers. Infection with a T. gondii clone showing low chicken virulence resulted in higher antibody levels in all chicken lines compared to infection with T. gondii clones of intermediate or high chicken virulence. This was in agreement with the parasite load as determined by real-time PCR. Overall, results show that progeny resulting from natural sexual recombination of T. gondii clonal lineages, may differ in their virulence for mice and chickens.

Seasonality in the proportions of domestic cats shedding Toxoplasma gondii or Hammondia hammondi oocysts is associated with climatic factors.

A previous study on domestic cats in Germany and neighbouring countries suggested seasonality in shedding Toxoplasma gondii oocysts. The aim of the present study was to elucidate whether this seasonality in shedding could be explained by climatic effects and whether differences between years in the proportions of cats shedding oocysts could also be explained by climatic factors. To this end, a long-term study over a period of 55 months on domestic cats for T. gondii and Hammondia hammondi oocysts was performed and the results compared with climatic data. Using species-specific PCR, T. gondii oocysts were identified in 0.14% (84/61,224) and H. hammondi in 0.10% (61/61,224) of the samples. Toxoplasma gondii oocysts were predominantly observed from summer to autumn, while H. hammondi oocysts were mainly found during autumn and winter. In statistical analyses using climatic data, even differences in parasitological findings between years could be partially modelled using monthly temperature, North Atlantic Oscillation indices and precipitation. Of the three climatic variables analysed, precipitation as an explanatory variable had the lowest impact in the statistical models while those taking only temperature and North Atlantic Oscillation indices into account were sufficiently predictive. Interestingly, time lags between the climatic event and the parasitological findings had to be implemented in all models. For T. gondii, North Atlantic Oscillation indices with a time lag of 7 months and temperature with a time lag of 2 months had the best predictive value. In contrast, temperature (with a time lag of 6 months) and the interaction of precipitation (with a time lag of 5 months) and North Atlantic Oscillation indices (with a time lag of 11 months) were optimal for predicting the seasonality of H. hammondi. These results suggest prominent differences in the life cycles of the two closely related parasites. Previous findings showed that H. hammondi lack avian hosts, in contrast to T. gondii, and the coincidence in the periods of high abundance of birds and high proportions of cats shedding T. gondii suggest that birds may play an important role in the epidemiology of this infection. The result that North Atlantic Oscillation index is an important variable in modelling variations in the proportion of cats shedding T. gondii and H. hammondi over the year is an indication that global warming may also influence the infection risk of animals and humans with T. gondii and H. hammondi. The findings have important implications for planning epidemiological studies and for estimating the risk of human infection.

Besnoitia besnoiti infection in cattle and mice: ultrastructural pathology in acute and chronic besnoitiosis.

Current knowledge on bovine besnoitiosis, caused by the emerging apicomplexan pathogen Besnoitia besnoiti, is still fragmentary. So far, studies dealing with ultrastructural pathology focused mainly on the easily accessible chronic stage, whereas ultrastructural investigations of tachyzoites were confined to in vitro studies. In the study presented here, the ultrastructural pathology of naturally B. besnoiti-infected cattle in the acute and chronic disease stages and experimentally B. besnoiti-infected mice was monitored. Further, the ultrastructure of tachyzoites and bradyzoites was investigated. Skin samples of two adult Limousin cows and one adult Limousin bull naturally infected with B. besnoiti and liver and skin samples of gamma-interferon knockout mice infected with B. besnoiti were examined in semithin sections stained with toluidine blue and safranin and in ultrathin sections contrasted with uranyl acetate and lead citrate. Samples of vessel walls of the bull and nasal mucosa of one cow were examined by scanning electron microscopy. Few tachyzoites-like endozoites were detected for the first time in bovine skin, and large numbers of tachyzoites were detected in murine skin and liver. Within tissue cysts in bovine skin, numerous bradyzoites were observed displaying signs of degeneration. Tachyzoites had apicomplexan endozoite ultrastructure. B. besnoiti tachyzoites and bradyzoites differed in shape and the number of amylopectin granules. Transmission and scanning electron microscopy confirmed the presence of two different cyst wall layers, and the present results on cyst wall ultrastructure were in accordance with those previously obtained by histological sections.

Genetic characterisation of Toxoplasma gondii isolates from European beavers (Castor fiber) and European wildcats (Felis silvestris silvestris).

Six free-ranging European beavers (Castor fiber) from Berlin greater metropolitan area and twelve European wildcats (Felis silvestris silvestris) originating from the German Federal State of Saxony-Anhalt were found dead and their carcasses were submitted for necropsy. Brain and lung samples were analysed for the presence of Toxoplasma gondii DNA. Histo-pathologic analysis of one beaver revealed several cyst-like protozoal structures in parts of the brain. Tissue DNA isolated from all animal samples was analysed by a specific T. gondii-PCR. Two beavers and four wildcats tested T. gondii-positive. DNA of the parasites was further analysed by PCR-RFLP typing using nine markers (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Only T. gondii type II alleles were detected, except for the Apico locus, where type I alleles were observed in two isolates from beavers and in three from wild cats. The results of this study indicate that type II T. gondii (including type II variant strain) is the most common genotype infecting wildcats and beavers from Germany.

Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs.

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p ≥ 0.05) and between IFAT and IB was moderate (κ=0.53, p ≥ 0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.

Toxoplasma gondii infection in sentinel and free-range chickens from Argentina.

This study aimed at isolating and genotyping Toxoplasma gondii from serologically positive free-range chickens from Argentina, and to evaluate the use of sentinel animals during a short time period of exposure to determine environmental contamination with T. gondii oocysts. Two groups of chickens on six farms were compared in this study: (i) young, 2-3 month-old broiler-type chickens reared as sentinel animals on the farms and (ii) adult chickens reared on the same farms for more than one year. Seroconversion rates of 7.0% or 5.7% were observed in sentinel broiler chickens reared for a period of 74 days (January-April 2010) or 88 days (August-November 2010) respectively, as shown by a T. gondii specific immunofluorescent antibody test. Fifty-three percent (17 of 32) of adult chickens were positive and showed higher titres than sentinel animals. Isolation of T. gondii from tissues (brain and heart) of serologically positive chickens was achieved from six of seven free-range adult birds with IFAT titres of 200 and higher. The isolated parasites were analysed by multi-locus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The isolated T. gondii showed three different genotypes: two genotypes consisted in atypical allele combinations, and the remaining genotype had exclusively clonal type II alleles. All isolates obtained at a single farm, corresponded to the same genotype. The T. gondii genotypes observed are identical to those described in cats, dogs, chickens and capybaras elsewhere in South America. Two isolates, which showed different allele combinations in PCR-RFLP, were characterized in a mouse virulence assay. While one isolate showed a low virulence a second isolate was of intermediate virulence to mice.

Toxoplasma gondii in foxes and rodents from the German Federal States of Brandenburg and Saxony-Anhalt: seroprevalence and genotypes.

Data on the genotypes of Toxoplasma gondii circulating in wildlife are scarce. In the present study, foxes and rodents from two Federal States in Central or Eastern Germany were examined for T. gondii infections. Body fluids were collected at necropsy or fluids were obtained from frozen tissues of naturally exposed red foxes (Vulpes vulpes), voles (Microtus arvalis), shrews (Neomys anomalus) and a striped field mouse (Apodemus agrarius) and tested for T. gondii by serology. DNA isolated from tissues of seropositive foxes and all the rodents was examined by PCR. In the German Federal States of Brandenburg and Saxony-Anhalt 152/204 (74.5%) and 149/176 (84.7%) of foxes, respectively, but none of the rodents (0/72) had antibodies to T. gondii. Only 28/152 (18.4%) and 20/149 (13.4%) of seropositive foxes from Brandenburg and Saxony-Anhalt, respectively, but none of the rodents tested PCR-positive for T. gondii. The complete T. gondii genotype could be determined for twelve samples using nine PCR-restriction fragment length polymorphism (PCR-RFLP) markers (newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico). In addition to T. gondii clonal type II (Apico II) and type II (Apico I), type III and T. gondii genotypes showing non-canonical allele patterns were observed in foxes. This suggests that, while T. gondii type II prevails in foxes, other genotypes circulate in wildlife. The population structure of T. gondii in Germany may be more diverse than previously thought.

Serological survey and risk factors for Toxoplasma gondii in domestic ducks and geese in Lower Saxony, Germany.

To obtain estimates for the prevalence of Toxoplasma gondii infection in ducks and geese in Germany, enzyme-linked immunosorbent assays (ELISA) were established based on affinity-purified T. gondii tachyzoite surface antigen 1 (TgSAG1) and used to examine duck and goose sera for T. gondii-specific antibodies. The results of 186 sera from 60 non-infected ducks (Anas platyrhynchos) and 101 sera from 36 non-infected geese (Anser anser) as well as 72 sera from 11 ducks and 89 sera from 12 geese inoculated experimentally with T. gondii tachyzoites (intravenously) or oocysts (orally) and positive in a T. gondii immunofluorescent antibody test (IFAT) were used to select a cut-off value for the TgSAG1-ELISA. Sera obtained by serial bleeding of experimentally inoculated ducks and geese were tested to analyze the time course of anti-TgSAG1 antibodies after inoculation and to assess the sensitivity of the assays in comparison with IFAT. In ducks, IFAT titres and ELISA indices peaked 2 and 5 weeks p.i with tachyzoites, respectively. Only three of six geese inoculated with tachyzoites at the same time as the ducks elicited a low and non-permanent antibody response as detected by the IFAT. In the TgSAG1-ELISA, only a slight increase of the ELISA indices was observed in four of six tachyzoite-inoculated geese. By contrast, inoculation of ducks and geese with oocysts led to an increase in anti-TgSAG1 antibodies within 1 or 2 weeks, which were still detectable at the end of the observation period, i.e. 11 weeks p.i. Inoculation of three ducks and three geese with oocysts of Hammondia hammondi, a protozoon closely related to T. gondii, resulted in a transient seroconversion in ducks and geese as measured by IFAT or TgSAG1-ELISA. Using the newly established TgSAG1-ELISA, sera from naturally exposed ducks and geese sampled in the course of a monitoring program for avian influenza were examined for antibodies to T. gondii; 145/2534 (5.7%) of the ducks and 94/373 (25.2%) of the geese had antibodies against TgSAG1. Seropositive animals were detected on 20 of 61 duck and in 11 of 13 goose farms; the seroprevalences within positive submissions of single farms ranged from 2.2% to 78.6%. Farms keeping ducks or geese exclusively indoors had a significantly lower risk (odds ratio 0.05, 95% confidence interval 0.01-0.3) of harboring serologically positive animals as compared with farms where the animals had access to an enclosure outside the barn.

Prevalence and genotypes of Toxoplasma gondii in feline faeces (oocysts) and meat from sheep, cattle and pigs in Switzerland.

The protozoan parasite Toxoplasma gondii infects almost all warm blooded animal species including humans, and is one of the most prevalent zoonotic parasites worldwide. Post-natal infection in humans is acquired through oral uptake of sporulated T. gondii oocysts or by ingestion of parasite tissue cysts upon consumption of raw or undercooked meat. This study was undertaken to determine the prevalence of oocyst-shedding by cats and to assess the level of infection with T. gondii in meat-producing animals in Switzerland via detection of genomic DNA (gDNA) in muscle samples. In total, 252 cats (44 stray cats, 171 pet cats, 37 cats with gastrointestinal disorders) were analysed coproscopically, and subsequently species-specific identification of T. gondii oocysts was achieved by Polymerase Chain Reaction (PCR). Furthermore, diaphragm samples of 270 domestic pigs (120 adults, 50 finishing, and 100 free-range animals), 150 wild boar, 250 sheep (150 adults and 100 lambs) and 406 cattle (47 calves, 129 heifers, 100 bulls, and 130 adult cows) were investigated by T. gondii-specific real-time PCR. For the first time in Switzerland, PCR-positive samples were subsequently genotyped using nine PCR-restriction fragment length polymorphism (PCR-RFLP) loci (SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) for analysis. Only one of the cats shed T. gondii oocysts, corresponding to a T. gondii prevalence of 0.4% (95% CI: 0.0-2.2%). In meat-producing animals, gDNA prevalence was lowest in wild boar (0.7%; 95% CI: 0.0-3.7%), followed by sheep (2.0%; 95% CI: 0.1-4.6%) and pigs (2.2%; 95% CI: 0.8-4.8%). The highest prevalence was found in cattle (4.7%; 95% CI: 2.8-7.2%), mainly due to the high prevalence of 29.8% in young calves. With regard to housing conditions, conventional fattening pigs and free-range pigs surprisingly exhibited the same prevalence (2.0%; 95% CI: 0.2-7.0%). Genotyping of oocysts shed by the cat showed T. gondii with clonal Type II alleles and the Apico I allele. T. gondii with clonal Type II alleles were also predominantly observed in sheep, while T. gondii with mixed or atypical allele combinations were very rare in sheep. In pigs and cattle however, genotyping of T. gondii was often incomplete. These findings suggested that cattle in Switzerland might be infected with Toxoplasma of the clonal Types I or III, atypical T. gondii or more than one clonal Type.

Atypical Toxoplasma gondii genotypes identified in oocysts shed by cats in Germany.

A total of 18,259 feline faecal samples from cats in Germany were collected and analysed for the presence of Toxoplasma gondii oocysts between June 2007 and December 2008. The proportion of T. gondii-positive samples collected between January and June was significantly lower than between July and December. The age of cats shedding T. gondii oocysts was not significantly different from the age of negative control cats. Forty-six T. gondii-positive samples were genetically characterised using nine PCR-restriction fragment length polymorphism (RFLP) markers which included newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. In addition, 22 isolates that had already been partially characterised in a previous study were further typed using PCR-RFLP markers c22-8, c29-2, L358, PK1 and Apico. Genotyping of the 68 isolates revealed that the majority of T. gondii isolates (n=54) had Type II patterns at all loci but displayed a Type I pattern at the Apico locus. Three isolates displayed Type II patterns at all loci, including the Apico locus. In addition, we detected one isolate with clonal Type III patterns at all loci and three isolates with atypical and mixed genotypes. Seven isolates could not be fully genotyped. One of those isolates displayed alleles of both Types I and II at the Apico locus. To our knowledge this is the first description of the presence of T. gondii genotypes different from the clonal Types I, II and III in the faeces of naturally infected cats.

First isolation of Neospora caninum from the faeces of a dog from Portugal.

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of gamma-interferon knockout mice with 10(2) sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.

Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers.

Neospora caninum infection is an important cause of bovine abortion. The infection can be transmitted transplacentally or by ingestion of oocysts shed by definitive hosts. There are few reports of dogs naturally shedding N. caninum oocysts and only some oocyst isolates were transferred into cell culture. The aim of the present study was to analyse N. caninum oocysts from the faeces of naturally infected dogs using a microsatellite-based typing technique and to compare them with cell culture-derived tachyzoites of the same isolates. To this end, N. caninum oocysts from six naturally infected dogs were inoculated into gamma-interferon knockout mice. After these mice had developed disease, tissue samples or peritoneal washings from necropsied mice were transferred into cell culture. Nested-PCR techniques were developed for the sensitive and specific amplification of N. caninum microsatellite-containing regions (MS1B, MS2, MS3, MS4, MS5 and MS10). DNA was extracted from oocysts and cell culture tachyzoites of each isolate, followed by amplification and sequence analysis of microsatellite-containing regions. Each parasite isolate examined yielded a unique microsatellite genotype, while no differences were revealed when data for N. caninum oocysts were compared with cultured tachyzoites of the same isolate. Our technique may allow the typing of clinical samples and different strains of N. caninum at the molecular level. This method may prove useful for the identification of infection sources in molecular epidemiological studies.

Characterization of a repetitive DNA fragment in Hammondia hammondi and its utility for the specific differentiation of H. hammondi from Toxoplasma gondii by PCR.

Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites. Both species use felids as definitive hosts and a broad spectrum of warm-blooded animals as intermediate hosts. Morphologically and serologically, the two parasites are difficult to differentiate. While T. gondii is an important pathogen of humans and a broad range of other vertebrates, disease has not yet been associated with H. hammondi infection. The aim of the present study was to identify and characterize a repetitive DNA fragment in H. hammondi and to evaluate its suitability for diagnostic purposes. With two primers considered to be specific for a 529 bp repetitive DNA fragment in T. gondii, weak products were amplified by polymerase chain reaction (PCR) from genomic DNA from H. hammondi oocysts. These amplicons (of approximately 150, 300 and 450 bp) were sequenced. The 292 bp consensus sequence of these three fragments revealed 84% identity with parts of the 529-bp repeat in T. gondii. Based on this sequence, a pair of primers was selected which amplified products of 98 and 630 bp from genomic DNA from H. hammondi oocysts but not from DNA from T. gondii. The 630-bp product was purified and cloned into a plasmid vector and the consensus sequence determined from seven randomly selected clones; comparison of this sequence with those available in current databases for T. gondii revealed an 84.0-88.1% identity over a length of 529 bp. The sequence data obtained was used for the development of a sensitive PCR which is entirely specific for H. hammondi and incorporates an internal control. The sequence data for the repetitive DNA element of H. hammondi provides a foundation for the design of primers specific to T. gondii, and the future optimisation of conventional and real-time PCR assays for the specific diagnosis of toxoplasmosis in definitive and intermediate hosts.

Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries.

Faecal samples of 24,106 cats from Germany and other European countries were examined microscopically in a veterinary laboratory in Germany between October 2004 and November 2006 to estimate the prevalence of animals shedding Toxoplasma gondii or Hammondia hammondi oocysts. Oocysts of 9-15 microm size with a morphology similar to that of H. hammondi and T. gondii were found in 74 samples (0.31%). A total of 54 samples were further characterised to achieve a species diagnosis and to determine the genotype of T. gondii isolates by PCR and PCR-RFLP. From these samples, 48 isolates were obtained: 26 (0.11%) were finally identified as T. gondii and 22 (0.09%) as H. hammondi. T. gondii-positive samples came from Germany, Austria, France and Switzerland while H. hammondi was detected in samples from Germany, Austria and Italy. In two samples (one T. gondii and one H. hammondi), PCR indicated the presence of Hammondia heydorni DNA. No Neospora caninum DNA was detected in any of the feline faecal samples. Twenty-two of the 26 T. gondii isolates could be genotyped. A PCR-RFLP analysis for the SAG2, SAG3, GRA6 and BTUB genes revealed T. gondii genotype II in all cases. Morphologically, H. hammondi oocysts exhibited a statistically significantly smaller Length-Width-Ratio than T. gondii oocysts.