Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife

Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.     

Toxoplasma gondii in sheep from the Campania region (Italy)

A cross-sectional serological survey was conducted in order to evaluate, irrespective of abortion, the Toxoplasma gondii infection in pastured sheep from the Campania region of southern Italy. A geographical information system was used in order to uniformly sample the ovine farms (n=117) throughout the entire region. Blood and milk samples were collected from 10 adult sheep (>18 months) on each farm (total number=1170 sheep). Serum samples were tested for the presence of IgG antibodies to T. gondii using a commercial indirect fluorescent antibody test. For each farm, the 10 milk samples collected were pooled in order to obtain a single milk sample per farm (total number=117 milk samples). The 77.8% (91/117) of the farms and the 28.5% (333/11,170) of the sheep resulted positive by serology. In addition, the presence of T. gondii DNA was detected by PCR in 4 milk samples out of the 117 examined (3.4%).     

Seroprevalence of Toxoplasma gondii in sows from slaughterhouses and in pigs from an indoor and an outdoor farm in Argentina

The objective of the present study was to determine the prevalence of Toxoplasma gondii antibodies from slaughter sows and from pigs raised at an indoor and an outdoor swine farm. Serum samples were obtained from 230 slaughter sows belonging to 83 farms distributed in 5 provinces. Blood samples were collected monthly from pigs of different ages from an intensive management indoor farm (farm 1). A cross-sectional study was carried-out from an outdoor farm (farm 2). All sera were tested for T. gondii antibodies by the modified agglutination test (MAT), using formalin-fixed tachyzoites as antigen. An antibody titer > or =1:25 was considered positive. Antibodies to T. gondii were detected in 87 (37.8%) of 230 sows sera. Distribution among provinces was: 37.1% from Santa Fe, 62.8% from Buenos Aires, 3.3% from San Luis, 58.7% from La Pampa and 24% from Córdoba. Four of 88 (4.5%) serum samples from farm 1 had antibodies to T. gondii and none of the negative pigs seroconverted. However, 45 of 112 samples from farm 2 were positive (40.2%) with the following distribution: sows 100%; nursery 40%; growers 13.8% and fatteners 20%. It is concluded that the prevalence of T.gondii antibodies among sows seems to be quite variable. T. gondii prevalence was related to the facilities and management of the farm.     

Toxoplasma gondii in invasive animals on the Island of Fernando de Noronha in Brazil: Molecular characterization and mouse virulence studies of new genotypes

This study aimed to genetically characterize and to determine virulence from Toxoplasma gondii samples from invasive animals in the Island of Fernando de Noronha, Brazil. Blood samples were collected from 21 tegu-lizard (Salvator merianae), 12 rock-cavies (Kerodon rupestris) and 154 black-rats (Rattus rattus) from the Island and MAT (cutoff 1:25) detected anti-T. gondii antibodies in 0% of the tegus (0/21); 58.3% of the rock-cavies (7/12) and 22.7% of rats (35/154). Tissue samples (brain, heart, liver and lung) from positive animals in MAT were collected for molecular analysis and for bioassay in Swiss Webster mice. After observation period, mice were euthanized, and serological detection and tissue cyst search in the brain were performed. The brain of positive animals for serological detection or tissue cyst search was cultured in MARC-145 cells for maintenance of the T. gondii isolate. No isolate was obtained from rock cavies. Nine isolates were obtained by bioassay of 35 seropositive black rats. DNA samples were extracted from rat tissues and from parasite isolates in cell culture, and genotyped using 10 PCR-RFLP markers. ToxoDB genotypes #78 (1) from rat tissue and #146 (4), #163 (2), #260 (2) and #291 (1) from cell culture were detected. Markers of genes ROP18 and ROP5 were analyzed and in vivo virulence test was conducted in mice. Analysis revealed two allele combinations, 3/1 and 3/3, indicating non-lethal T. gondii strains, which is supported by mouse virulence test.     

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.     

High prevalence and genotypes of Toxoplasma gondii isolated from organic pigs in northern USA

The ingestion of undercooked pork infected with Toxoplasma gondii is considered an important source of transmission of this parasite. While T. gondii infection in confinement raised market pigs (market pigs are typically used for fresh, unprocessed pork products) in the USA has decreased significantly over the last 20 years, infection levels in pigs with access to the outdoors can be quite high. An upsurge in consumer demand for 'organically raised', 'humanely raised' and 'free range' pork products has resulted in increasing numbers of hogs being raised in non-confinement systems. To determine T. gondii infection rate in these organic pigs, prevalence of T. gondii in organically raised pigs in two establishments (Farm 1, Farm 2) in Michigan was investigated. Serum and tissue samples from 33 pigs on the farm were available for T. gondii evaluation at slaughter. Serological testing was performed using both ELISA and the modified agglutination test (MAT). Antibodies to T. gondii were detected by both ELISA and MAT in 30 of 33 animals with MAT titers of 1:25 in three, 1:50 in six, 1:100 in seven, 1:200 in 13, and 1:400 in one. Hearts of all 33 pigs were bioassayed for T. gondii in mice; T. gondii was isolated from 17 pigs including one from a seronegative (both ELISA and MAT) pig. Genetic typing of 16 of the 17 T. gondii isolates using the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico loci revealed clonal Type II from Farm 1 and clonal Type III on Farm 2. These results revealed very high prevalence of T. gondii in organic pigs for the first time in USA, indicating potentially increased health risk of consuming organic swine products.     

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC(50) of 2.5 μM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.     

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.     

Isolation and genetic characterization of Toxoplasma gondii from alpaca (Vicugna pacos) and sheep (Ovis aries)

Alpacas are important to the economy of several countries. Little is known of Toxoplasma gondii infection in alpacas worldwide. In the present study, T. gondii was isolated and genetically characterized from alpacas for the first time. Alpacas (n?=?16) and rams (n?=?12) pastured on a farm in Virginia, USA, were examined at necropsy. Antibodies to T. gondii were determined by the modified agglutination test (MAT, 1:25) and found in 6 of 16 alpacas with titers of 1:100 (2 alpaca), 1:400 (2 alpacas), 1:800 (1 alpaca), and 1:1,600 (1 alpaca), and 5 of 12 rams in titers of 1:50 in one, 1:400 in one, 1:800 in one, 1:1,600 in one, and 1:3,200 in one. Tissues of all 16 alpacas were bioassayed in mice or in cats. Muscles (heart, skeletal muscle) of nine alpacas with MAT titers of 1:25 were fed to T. gondii-free cats; the cats did not shed oocysts. Viable T. gondii was isolated from tissues of two of six seropositive alpacas by bioassay in mice. Viable T. gondii was isolated from three of three seropositive sheep by bioassay in mice. Genotyping using cell-cultured tachyzoites revealed four genotypes, including one for ToxoDB PCR-RFLP genotype #2 (type III), one for genotype #3 (type II variant), one for genotype #170, and two for a new genotype designated as ToxoDB PCR-RFLP genotype #230. Thus, four of the five T. gondii isolates in the present study belonged to different genotypes. These results indicate a higher genetic diversity among T. gondii isolates circulating in the USA than previously realized.     

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.     

Seroprevalence of Toxoplasma gondii infection in goats and sheep in Zimbabwe

Seroprevalence rates of Toxoplasma gondii anti-antibodies in adult goats and sheep from different parts of Zimbabwe were determined. A total of 225 (67.9%) of the 335 serum samples tested were positive for anti-T. gondii IgG antibodies with the indirect fluorescent antibody test. There were differences in antibody seroprevalences among communal land goats from the different agro-ecological zones (Natural regions llb and III: 80 and 96.7%, respectively; Natural region IV: 65.9%; Natural region V: 45%; and Natural region III had a significantly higher seroprevalence than IV and V. The highest seroprevalences found in Natural regions II b and Ill are likely to be linked to the existence of more households and hence the possibility of a higher concentration of domestic cats that increases the chances of environmental contamination with their faeces harbouring T. gondii oocysts. The seroprevalence rate in sheep from a large commercial farm (10%) was significantly lower than that of sheep reared under the communal grazing system (80%). Overall, significantly higher proportions of seropositive animals had antibody titres of 1:50 (34.2% of 225) and 1:100 (44% of 225) as compared to the 9.8% and 12% with antibody titres of 1:200 and > or =1:400, respectively.     

Immunohistochemical identification of Toxoplasma gondii in tissues from Modified Agglutination Test positive sheep

Toxoplasma gondii is a zoonotic agent of great importance in veterinary and public health. The aim of this study was to identify T. gondii by IHC (immunohistochemistry) in different sheep tissues and to determine if an association exists between the results obtained by this method and those obtained by the Modified Agglutination Test (MAT). Tissue specimens of twenty-six sheep seroreactive for T. gondii were selected for histopathological evaluation. The presence of T. gondii was investigated in brain, liver and heart samples by IHC and a possible anti-T. gondii antibody cross reactions with other parasites. McNemar's, Chi-square and Fisher's Exact Tests were applied for the statistical analysis of the results. The analysed tissues showed at least one of the following histopathological changes: mild-to-moderate congestion, focal polymorphonuclear inflammatory infiltrate and multifocal or focal mononuclear inflammatory infiltrate. Sarcocystis spp. were identified in the histological sections from both the heart and diaphragm tissues of 88.5% (23/26) of the animals. A total of 46.2% (12/26) of the T. gondii seroreactive sheep was also positive for T. gondii by IHC in at least one organ (brain, liver or heart). The liver IHC-positivity for T. gondii was statistically equivalent to the global individual IHC-positivity, according to McNemar's test. In addition, IHC allowed the detection of T. gondii in infected animals regardless of the titration observed in the MAT. The statistical difference observed between the three organs when comparing the low titration group, suggested that the heart might be the most suitable organ to detect T. gondii infection by IHC. The IHC results in this study revealed that almost half of MAT positive animals could serve as potential sources of infection for humans because bradyzoites were identified in different tissues, regardless of the MAT titration.     

Isolation and molecular characterization of Toxoplasma gondii from chickens and ducks from Egypt

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 121 free range chickens (Gallus domesticus) and 19 ducks (Anas sp.) from a rural area surrounding Giza, Egypt was assessed. Blood, heart, and brain from each animal were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 49 (40.4%) chickens in titers of 1:5 in 11, 1:10 in four, 1:20 in four, 1:40 in eight, 1:80 in 10, and 1:160 or more in 12 chickens. Antibodies were found in three ducks each with a titer of 1:80. Hearts and brains of seropositive (MAT > or = 1:5) chickens and ducks were bioassayed in mice. Additionally, hearts and brains of seronegative (MAT<1:5) animals were bioassayed in T. gondii-free cats. T. gondii was isolated from 19 of 49 seropositive chickens (one with a titer of 1:5, two with a titer of 1:20, one with a titer of 1:40, five with a titer of 1:80, three with a titer of 1:160, and seven with a titer of > or = 1:360). One cat fed tissues pooled from 15 seronegative chickens shed T. gondii oocysts, while two cats fed tissues of 34 seronegative chickens did not shed oocysts. T. gondii was isolated from one of the seropositive ducks by bioassay in mice. The two cats fed tissues from 16 seronegative ducks did not shed oocysts. Genotyping of 20 chicken isolates of T. gondii using the SAG 2 locus indicated that 17 isolates were type III and three were type II. The duck isolate of T. gondii was type III. The mice inoculated with tissue stages of all 21 isolates of T. gondii from chickens and ducks remained asymptomatic, indicating that phenotypically they were not type I because type I strains are lethal for mice. Infections with mixed genotypes were not found.     

Genetic and biologic characterization of Toxoplasma gondii isolates of cats from China

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 34 cats from People's Republic of China. Antibodies to T. gondii were assayed by the modified agglutination test and found in 27 of 34 (79.4%) cats with titers of 1:40 in one, 1:80 in one, 1:160 in three, 1:320 in three, 1:640 in eight, and 1:1280 or higher in 11 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, and tongue) of 27 seropositive cats were pooled and bioassayed in mice (8 cats) or cats (19 cats). T. gondii was isolated from tissues of 17 of 27 seropositive cats. Genotyping of these 17 T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and a new SAG2, and an apicoplast marker Apico revealed two genotypes. This is the first report of genetic typing of T. gondii isolates from cats from China.     

Toxoplasma gondii infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping by microsatellite analysis

Ingesting meat of free-range livestock, mainly sheep, is associated with human toxoplasmosis in European countries. Data on Toxoplasma gondii infection in French ovine livestock are relatively scarce. Sera from 164 lambs and 93 ewes slaughtered in Haute-Vienne district, France, were tested by a direct agglutination test. Antibodies to T. gondii were found in 36 (22.0%) lambs and in 61 (65.6%) ewes. In addition, to attempt parasite isolation for genotyping, hearts from 50 other ewes were obtained from a local slaughterhouse, and were screened by a direct agglutination test. T. gondii was isolated in 8 of 30 seropositive hearts bioassayed in mice. All isolates were type II by genetic characterization at five microsatellite loci (TUB2, TgM-A, W35, B17, B18). These results indicate that bovines slaughtered in France may be highly infected by T. gondii with a potential risk of parasite transmission to humans by consumption of undercooked meat. Multilocus microsatellite analysis shows the predominance of type II in sheep as previously described in humans.     

A serological study on the prevalence of Toxoplasma gondii in sheep of Lithuania

In the present study the seroprevalence of the protozoan parasite Toxoplasma gondii infection in sheep was investigated in 6 regions of Lithuania. Blood samples were taken from 354 sheep and were tested using commercial ELISA method. The total seroprevalence of Toxoplasma gondii infection in sheep was 42.1%. Significant differences in seroprevalence were observed between age groups (P < or = 0.05). The results of this investigation suggest that the Toxoplasma gondii parasite is widely spread, and can be one of reasons of sheep abortion in Lithuania.     

Toxoplasma gondii: level of carriage in sheep of Marrakech region (Mnabha)

Toxoplasma gondii is an ubiquitous parasite with a prevalence variable from country to country. In Morocco very few studies were devoted to this prevalence. To fill this gap we were interested to study the epidemiology of this parasite and to know the level of carriage by the different vectors which are the sources of contamination in humans. The study was done by directly detecting the cysts in the cerebral tissue of the 50 sheep killed and destined for consumption. The results of this preliminary study show that 30% of the cases carry the cysts of T. gondii. To confirm this result and verify the virulence, cerebral specimens were inoculated into mice. These findings are encouraging to complete this study with serological tests and to look for the parasite in cows and goats of this region.