Age-related changes in cerebral congenital toxoplasmosis: Histopathological and immunohistochemical evaluation.

Congenital toxoplasmosis is a widespread worldwide disease producing varying degrees of damage to the fetus including ocular and neurological impairment. However, the underlying mechanisms are not yet clear. Therefore, the current study aimed to investigate the progress of congenital cerebral toxoplasmosis in experimentally infected offspring animal model at different age groups till become adults. To fulfill this aim, the offspring of Me49 T. gondii infected pregnant mice were divided into groups; embryo, infant, young and adult phases. Blood and brain samples were collected for further hormonal and histopathological studies and immunohistochemical staining of glial fibrillary acidic protein (GFAP) and synaptophysin (SYN). Our results showed several encephalitic changes in the infected groups ranging from gliosis to reduced cortical cell number and fibrinoid degeneration of the brain. We showed increased expression of GFAP and SYN indicating activation of astrocytes and modification of the synaptic function, respectively. These changes started intrauterine following congenital infection and increased progressively afterward. Moreover, infected mice had elevated corticosterone levels. In conclusion, the current study provided new evidences for the cellular changes especially in the infected embryo and highlighted the role of GFAP and SYN that may be used as indicators for T. gondii-related neuropathy.

Adjunctive bradyzoite-directed therapy for reducing complications of congenital toxoplasmosis.

Congenital toxoplasmosis is caused by in utero infection of the fetus with the intracellular parasite Toxoplasma gondii. Upon infection, the parasite forms life-long cysts in fetal brain and eyes which are resistant to the currently accepted therapy of pyrimethamine and sulfadiazine. These cysts commonly reactivate later in life causing chorioretinitis and visual impairment, and rarely cause neurological complications. I hypothesize that adjunctive, bradyzoite-directed therapies have the potential to alleviate a significant burden of disease by reducing cyst burden in neonatal brain and eyes. Atovaquone is perhaps the most promising drug for further evaluation given its low side-effect profile, established safety, and efficacy in animal models reducing cyst burden. Very limited observational data in humans suggests atovaquone may prevent Toxoplasma-associated chorioretinitis recurrence. Clinical trials are needed to evaluate it and other potential drugs as adjunctive treatment in congenital toxoplasmosis.

Detection and genotypic characterization of Toxoplasma gondii DNA within the milk of Mongolian livestock.

Toxoplasma gondii is a global, zoonotic parasite capable of infecting any warm-blooded host. Toxoplasmosis can cause a variety of illnesses including abortions and congenital defects in humans, sheep, and goats. Congenital toxoplasmosis is considered to have the highest global disease burden of any foodborne illness in humans. This study examined the potential role of milk as a route of T. gondii transmission between livestock and humans within Mongolian herders, a little-studied population which relies heavily on animals. Milk of Mongolian sheep, goats and Bactrian camels was tested for the presence of T. gondii DNA, and a survey was conducted to ascertain what behavioral and environmental factors were present that might potentiate T. gondii infection within these Mongolian communities. T. gondii DNA was detected in samples from one sheep and five camels. Sequence analysis of DNA from camel milk revealed that two were from potentially virulent T. gondii genotypes. This has implications for public health in the region, as milk is an extremely important source of nutrition and our survey results imply that some people believe consumption of raw camel milk carries health benefits. This is the first report of T. gondii DNA in Bactrian camel milk as well as the first genotypic characterization of T. gondii within Mongolia.

Vertical transmission and fetal damage in animal models of congenital toxoplasmosis: A systematic review.

In humans, the probability of congenital infection and fetal damage due to Toxoplasma gondii is dependent on the gestation period at which primary infection occurs. Many animal models have been used for vaccine, drug testing, or studies on host or parasite factors that affect transmission or fetal pathology, but few works have directly tested fetal infection and damage rates along gestation. So, the purpose of this work was to perform a systematic review of the literature to determine if there is a model which reflects these changes as they occur in humans. We looked for papers appearing between 1970 and 2014 in major databases like Medline and Scopus, as well as gray literature. From almost 11,000 citations obtained, only 49 papers fulfilled the criteria of having data of all independent variables and at least one dependent datum for control (untreated) groups. Some interesting findings could be extracted. For example, pigs seem resistant and sheep susceptible to congenital infection. Also, oocysts cause more congenitally infected offspring than tissue cysts, bradyzoites or tachyzoites. In spite of these interesting findings, very few results on vertical transmission or fetal damage rates were similar to those described for humans and only for one of the gestation thirds, not all. Moreover, in most designs tissue cysts - with unknown number of bradyzoites - were used, so actual dose could not be established. The meta-analysis could not be performed, mainly because of great heterogeneity in experimental conditions. Nevertheless, results gathered suggest that a model could be designed to represent the increase in vertical transmission and decrease in fetal damage found in humans under natural conditions.

Mouse model of congenital infection with a non-virulent Toxoplasma gondii strain: Vertical transmission, "sterile" fetal damage, or both?

Congenital transmission of Toxoplasma gondii may occur if the mother gets infected for the first time while pregnant. The risk of mother-to-child transmission depends on the gestation trimester at infection, being lowest in the first and highest in the last. Conversely, fetal damage is frequent and more severe at the beginning of pregnancy. The objective of this study was to evaluate congenital transmission and pathological aspects in the placenta and the fetus using a mouse model of congenital infection of the second gestation third. Forty-five female BALB/c mice were infected intravenously with 2.5-10.0 × 10(6) tachyzoites of the ME49 strain at middle gestation. Samples of maternal spleen and fetal/placental units were taken 72 h later. We determined parasite load and vertical transmission by qPCR, as well as damage macroscopically and by histopathology. With the lowest dose, 18% of the fetuses were infected. Also, 40% of fetuses/litter were altered, while this value was 10% in the control group (P < 0.05). These results are similar to those described in humans in terms of vertical transmission and fetal damage during the second third of gestation. The maternal spleen had 10-1000 times more tachyzoites than the placenta, and the later retained 90-99% of the parasites that could reach the fetus. Nevertheless, we found resorptions, abortions or fetal tissue damage in the presence but also in the absence of parasites. Our data indicate a strong protective effect of maternal organs and the placenta against fetal infection, but extensive damage of the later may led to resorption or abortion without vertical transmission.

Anti-Toxoplasma Activity of Estragole and Thymol in Murine Models of Congenital and Noncongenital Toxoplasmosis.

Toxoplasmosis is caused by Toxoplasma gondii , an obligatory intracellular protozoan. Normally benign, T. gondii infections can cause devastating disease in immunosuppressed patients and through congenital infection of newborn babies. Few prophylactic and therapeutic drugs are available to treat these infections. The goal of the present study was to assess the anti-Toxoplasma effects in a congenital and noncongenital model of toxoplasmosis (using ME49 strain), besides assessing immunological changes, in vitro cytotoxicity, and in vivo acute toxicity of commercial estragole and thymol. The congenital experimental model was used with intermediate stages of maternal infection. The serum levels of immunoglobulin (Ig)M, IgG, interleukin (IL)-10, IL-12, and interferon-gamma (IFN-γ) were quantified from infected and treated C57Bl/6 mice. Estragole and thymol respectively exhibited low to moderate in vivo toxicity and cytotoxicity. Animals treated with estragole showed high IFN-γ and strong type 1 helper T cell response. Both compounds were active against T. gondii ME49 strain. Furthermore, orally administered estragole in infected pregnant mice improved the weight of offspring compared with untreated controls. Subcutaneous administration of both compounds also increased the weight of mouse offspring born to infected mothers, compared with untreated controls. Estragole and thymol display important anti-Toxoplasma activity. Further studies are needed to elucidate the mechanism of action of these compounds.

Dual congenital transmission of Toxoplasma gondii and Sarcocystis neurona in a late-term aborted pup from a chronically infected southern sea otter (Enhydra lutris nereis).

Toxoplasma gondii and Sarcocystis neurona are protozoan parasites with terrestrial definitive hosts, and both pathogens can cause fatal disease in a wide range of marine animals. Close monitoring of threatened southern sea otters (Enhydra lutris nereis) in California allowed for the diagnosis of dual transplacental transmission of T. gondii and S. neurona in a wild female otter that was chronically infected with both parasites. Congenital infection resulted in late-term abortion due to disseminated toxoplasmosis. Toxoplasma gondii and S. neurona DNA was amplified from placental tissue culture, as well as from fetal lung tissue. Molecular characterization of T. gondii revealed a Type X genotype in isolates derived from placenta and fetal brain, as well as in all tested fetal organs (brain, lung, spleen, liver and thymus). This report provides the first evidence for transplacental transmission of T. gondii in a chronically infected wild sea otter, and the first molecular and immunohistochemical confirmation of concurrent transplacental transmission of T. gondii and S. neurona in any species. Repeated fetal and/or neonatal losses in the sea otter dam also suggested that T. gondii has the potential to reduce fecundity in chronically infected marine mammals through parasite recrudescence and repeated fetal infection.

A rare case of feline congenital Toxoplasma gondii infection: fatal outcome of systemic toxoplasmosis for the mother and its kitten.

This report describes a case of fatal systemic toxoplasmosis in a 2.5-year-old mixed breed pregnant cat and its kittens. The pregnant cat was presented to the gynecology clinic with symptoms of dystocia. The ultrasound examination revealed the presence of five fetuses in the uterus, three of which were not alive, and consequently a cesarean section was performed. However, the mother cat and the remaining two live kittens died two and ten days after cesarean section, respectively. Pathologically, severe alveolar edema, tachyzoite-like structures in the alveolar macrophages and multifocal necroses in the lungs of mother cat were observed. An intense Toxoplasma gondii immunopositive reaction was observed in the cytoplasms of alveolar macrophages, bronchial and bronchiolar epithelia, necrotic foci in the lungs, and Kupffer cells of the liver. PCR analyses amplified T. gondii DNA in tissue samples of the mother cat and kittens. The present study provides strong evidence for a transplacental transmission of T. gondii infection with deadly outcome for the mother cat, fetuses and kittens. As to the authors' knowledge, this report is the first case of fatal congenital toxoplasmosis in domestic cats in Turkey.

Toxoplasma gondii abortion storm in sheep on a Texas farm and isolation of mouse virulent atypical genotype T. gondii from an aborted lamb from a chronically infected ewe.

Sheep are commonly infected with the protozoan parasite, Toxoplasma gondii. Infection may cause early embryonic death and resorption, fetal death and mummification, abortion, stillbirth, and neonatal death. Most sheep acquire T. gondii infection after birth. Recent studies reported that congenital ovine transmission of T. gondii may be more common than previously believed, but these findings are solely based on PCR data and require confirmation using other techniques to verify the findings. In the present study, during the lambing season of 2005 a toxoplasmosis abortion storm occurred in a flock of purebred Suffolk ewes on a farm in Texas. Only 14 healthy lambs were born, and 38 abortuses, mummies and weak or stillborn lambs were delivered. Another 15 fetuses identified by ultrasound were presumably resorbed or were aborted undetected. Antibodies to T. gondii were found in 37 (94.8%) of the 39 ewes and 30 of them had high titers (1:3200 or higher) when tested in the modified agglutination test (MAT). In the 2006 lambing season, two (both with MAT titers of ≥ 3200 in 2005) of 26 ewes delivered T. gondii infected lambs. T. gondii tissue cysts were found histologically in lesions of encephalitis in a lamb from one ewe and viable T. gondii (designated TgShUs55) was isolated from the brain and heart of a lamb from the second ewe. TheTgShUs55 had an atypical genotype using 10 PCR-RFLP markers, and was 100% lethal for Swiss Webster mice, irrespective of the dose or the stage of the parasite inoculated. In subsequent seasons, the ewes lambed normally. The results of the present study support the hypothesis that most sheep that have aborted due to T. gondii develop protection against future toxoplasmosis induced abortion, but the protection is not absolute.

Congenital toxoplasmosis in wild boar (Sus scrofa) and identification of the Toxoplasma gondii types involved.

Congenital toxoplasmosis has been little described in wild animals. We report a case of vertical transmission in wild boar (Sus scrofa). Necropsy and histopathologic examination of a pregnant female and her three fetuses revealed all to have lesions compatible with acute toxoplasmosis. Nested polymerase chain reaction B1 gene detected Toxoplasma gondii in maternal (heart and diaphragm) and fetal (central nervous system, retina, optic nerve, heart, lung, tongue, and diaphragm) samples. The mother had a mixed infection of T. gondii types I and III. One fetus with type III infection developed no malformations, but the others-one with type I infection and one infected by types I and III-showed bilateral ocular agenesis, prognathism, and agenesis of the nasal cartilage. These results suggest the pathogenicity of the various T. gondii types may differ in wild boars.

Congenital infection of mice with Toxoplasma gondii induces minimal change in behavior and no change in neurotransmitter concentrations.

We examined the effect of maternal Toxoplasma gondii infection on behavior and the neurotransmitter concentrations of congenitally infected CD-1 mice at 4 and 8 wk of age when latent tissue cysts would be present in their brains. Because of sex-associated behavioral changes that develop during aging, infected female mice were compared with control females and infected male mice were compared with control males. Only the short memory behavior (distance between goal box and first hole investigated) of male mice congenitally infected with T. gondii was significantly different (P < 0.05) from that of uninfected control males at both 4 and 8 wk by using the Barnes maze test. The other parameters examined in the latter test, i.e., functional observational battery tests, virtual cliff, visual placement, and activity tests, were not significantly different (P > 0.05) at 4 and 8 wk. Concentrations of neurotransmitters and their metabolites (dopamine; 3,4-dihydroxyphenylacetic acid; homovanillic acid; norepinephrine; epinephrine; 3-methoxy-4-hydroxyphenylglycol; serotonin; and 5-hydroxyindoleacetic acid) in the frontal cortex and striatum were not different (P > 0.05) between infected and control mice at 8 wk of age. The exact mechanism for the observed effect on short-term memory in male mice is not known, and further investigation may help elucidate the molecular mechanisms associated with the proposed link between behavioral changes and T. gondii infection in animals. We were not able, however, to confirm the widely held belief that changes in neurotransmitters result from chronic T. gondii infection of the brain.

Protection in a hamster model of congenital toxoplasmosis.

Almost uniform protection against congenital toxoplasmosis initiated by inoculations with cysts and oocysts of the parasite was seen in the hamster model, among strains of different genotypes. Because the RH immunization prior to pregnancy has to be controlled with medication for most of the hamsters to survive, and also some congenital transmission of Toxoplasma was observed during the chronic stage of the infection, the hamster is considered less practical than the rat and the BALB/c mouse models. It is concluded that the hamster model closely resembles protection against congenital infection in nature, where most of the pregnant women and ewes that experienced a toxoplasma infection previously, protect their fetuses against an infection with the parasite during pregnancy.

Toxoplasma gondii: infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts.

Two studies, of a natural infection and an experimental infection, were performed in order to study congenital transmission of Toxoplasma gondii in cattle. In the first study, 50 fetuses were harvested from gestating cows that were eutanasied at a municipal slaughterhouse in Jaboticabal, São Paulo state, Brazil. In the second study, 11 gestating cows were divided into four groups for inoculation with T. gondii: GI consisted of three cows inoculated with 1.0 × 10(5) oocysts during their first trimester of gestation; GII consisted of three cows inoculated with 1.0 × 10(5) oocysts during their second trimester of gestation; GIII consisted of three cows inoculated with 1.0 × 10(5) oocysts during their last trimester of gestation; and GIV consisted of two control cows, one during its first and the other during its second trimester of gestation. In both studies, the presence of T. gondii was confirmed both indirectly by immunofluorescence assay (IFAT). In the natural infection experiment, 18% (9/50) of the gestating cows were confirmed to have specific antibodies (IFAT--1:64) against T. gondii. The bioassay was able to diagnose the presence of T. gondii in the tissue samples from three calves. In the second experiment, the nine cows from groups I, II and III presented with specific antibodies (IFAT) against T. gondii. In contrast, T. gondii could not be detected by IFAT, histopathological examination or the bioassay in any of the nine calves born to cows experimentally infected with T. gondii oocysts. Based on the results from both studies, we conclude that congenital infection of T. gondii in cattle, while infrequent, does occur naturally. The pathogenicity of the strain of T. gondii may influence the likelihood of this route of transmission.

Evaluation of vertical transmission of Toxoplasma gondii in Calomys callosus model after reinfection with heterologous and virulent strain.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes a variety of clinical syndromes, but the infection is severe in immunocompromised individuals and during pregnancy due to the possibility of transplacental transmission of the parasite causing congenital toxoplasmosis. Vertical transmission of the parasite usually occurs when females are primarily infected during pregnancy. Calomys callosus is resistant to T. gondii ME49 strain, which presents a moderate virulence and congenital disease occurs only during the acute phase of infection. The aim of this study was to determine whether vertical transmission occurs when females of C. callosus chronically infected with ME49 strain of T. gondii are reinfected with a highly virulent strain (RH, type I). Females were infected with cysts of the ME49 strain. On the 1st day of pregnancy, animals were reinfected with tachyzoites of the RH strain. In the 19th day of pregnancy, placentas and embryos were processed for morphological analysis, immunohistochemistry and for detection of the parasite by PCR and mouse bioassay. Morphological and immunohistochemical analyses revealed the presence of parasites only in placental tissues. Mouse bioassay results showed seroconversion only in mice that were inoculated with placental tissues. Also, T. gondii DNA was detected only in placental samples. Congenital toxoplasmosis does not occur in C. callosus females chronically infected with the moderately virulent ME49 strain of T. gondii and reinfected with the highly virulent RH strain, thus indicating that primary T. gondii infection before pregnancy leads to an effective long-term immunity preventing transplacental transmission to the fetus.

Congenital transmission of Toxoplasma gondii in deer mice (Peromyscus maniculatus) after oral oocyst infection.

To investigate how different routes of Toxoplasma gondii transmission influence the antibody response and infection status of deer mice (Peromyscus maniculatus), 80 mice were orally infected with 1, 5, 10, or 100 T. gondii oocysts. Ten weeks postinfection, 15 T. gondii -seropositive female mice were bred and allowed to produce 2 litters. Evidence of persistent T .gondii infection in orally infected mice was detected by serology and DNA amplification in mice from all 4 oocyst treatment groups, including those that received only a single T. gondii oocyst. Congenital transmission of T. gondii was detected by polymerase chain reaction (PCR) in 7/8 first and 4/7 second litters. Toxoplasma gondii was also detected by PCR in 9/30 congenitally infected offspring 16 wk after birth, despite the fact that detectable serological titers had waned. These findings raise questions about the applicability of serological testing to assess the prevalence of T. gondii infection in deer mice and other rodents in the wild. Additionally, the detection of frequent congenital transmission suggests that deer mice could help maintain T. gondii in the environment even in the absence of definitive feline hosts.

A serological study and subsequent isolation of Toxoplasma gondii from aborted ovine fetuses in Mashhad area, Iran.

Toxoplasmosis is an important cause of abortion and stillbirth in sheep on a worldwide basis. In the present study, the role of Toxoplasma gondii in inducing abortion in sheep of the Mashhad area of Iran was addressed using serological and parasitological methods. In total, 325 aborted ovine fetuses were collected between 2006 and 2008 during lambing season. Thoracic and abdominal fluids of aborted fetus were serologically investigated with a T. gondii -IFAT. Antibody titers equal to or greater than 1:20 were detected in 17 (5.2%) of ovine fetuses. Processed brain samples of seropositive ovine fetuses were intraperitoneally inoculated into mice for isolation of T. gondii . The process yielded a single T. gondii isolate that remained avirulant for mice after several passages. The results document that T. gondii is among the important causes of ovine abortion in Iran.

Murine neonatal infection provides an efficient model for congenital ocular toxoplasmosis.

Congenital infection is one of the most serious settings of infection with the apicomplexan parasite Toxoplasma gondii. Ocular diseases, such as retinochoroiditis, are the most common sequels of such infection in utero. However, while numerous studies have investigated the physiopathology of acquired toxoplasmosis, congenital infection has been largely neglected so far. Here, we establish a mouse model of congenital ocular toxoplasmosis. Parasite load and ocular pathology have been followed for the first 4 weeks of life. Ocular infection developed slowly compared to cerebral infection. Even after 4 weeks, not all eyes were infected and ocular parasite load was low. Therefore, we evaluated a scheme of neonatal infection to overcome problems associated with congenital infection. Development of infection and physiopathology was similar, but at a higher, more reliable rate. In summary, we have established a valuable model of neonatal ocular toxoplasmosis, which facilitates the research of the underlying physiopathological mechanisms and new diagnostic approaches of this pathology.

Evaluation of Toxoplasma gondii placental transmission in BALB/c mice model.

Toxoplasma gondii infection is common worldwide and highly important to pregnant women as it can be transmitted to the fetus via the placenta. This study aimed at evaluating the prevention of placental transmission in two different strains after chronic infection with each one of the strains. A BALB/c mice model was inoculated 30days before breeding (immunization) and re-infected 12 and 15days after pregnancy (challenge). Seven experimental groups were assayed: G1: ME49-immunization (type II), M7741-challenge (type III); G2: M7741-immunization, ME49-challenge; G3, ME49-immunization; G4: M7741-immunization; G5: ME49-challenge; G6: M7741-challenge; G7: saline solution inoculation. Serology, mouse bioassay, PCR and RLFP of the uterus, placenta and fetus were performed to determine the congenital transmission of the strains challenged after chronic infection. IgG T. gondii antibodies were detected in G1, G2, G3 and G4, but not in G5, G6 and G7. All animals of G5 and G6 were IgM-positive. Congenital infection was not detected by bioassay and PCR. Nonetheless, placentas from G3 and G4 resulted positive but no corresponding fetal infection was detected. G1 and G2 did not show the genotype of the strain challenged during pregnancy, only those of chronic infection. Thus, the chronically infected BALB/c mice showed no re-infection after inoculation with another strain during pregnancy. Further studies with different parasite loads and different mice lineages are needed.

Veterinary vaccines against Toxoplasma gondii.

Toxoplasma gondii has a very wide intermediate host range and is thought to be able to infect all warm blooded animals. The parasite causes a spectrum of different diseases and clinical symptoms within the intermediate hosts and following infection most animals develop adaptive humoral and cell-mediated immune responses. The development of protective immunity to T. gondii following natural infection in many host species has led researchers to look at vaccination as a strategy to control disease, parasite multiplication and establishment in animal hosts. A range of different veterinary vaccines are required to help control T. gondii infection which include vaccines to prevent congenital toxoplasmosis, reduce or eliminate tissue cysts in meat producing animals and to prevent oocyst shedding in cats. In this paper we will discuss some of the history, challenges and progress in the development of veterinary vaccines against T. gondii.