Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii.

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8­11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.

Comparative virulence of Caribbean, Brazilian and European isolates of Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a zoonotic parasite of global importance. The outcome of infection in humans can depend on a number of factors including the infecting stage of the parasite, inoculating dose and virulence of the infecting strain. Molecular epidemiological studies have demonstrated an abundance of atypical strains of T. gondii in South America, many of which have been associated with more severe sequelae of infection. The aim of this study was to compare the virulence of T. gondii strains isolated in the Caribbean to a virulent Brazilian strain and an avirulent European strain. METHODS: One hundred and twenty Swiss CD-1 mice were split into 8 groups of 15 mice and each group was inoculated with 200 tachyzoites of one of 8 isolates, comprising ToxoDB genotypes #1, #141, #265, #13, #3 and #6. Five mice per group were euthanized at day 8 post-inoculation (p.i.) and parasite burden was determined in heart, lungs and eyes using quantitative PCR. Lungs and brain were also examined by histopathology and immunohistochemistry. The remaining 10 mice per group were part of a survival experiment to assess virulence. DNA was extracted from tachyzoites of each of the 8 T. gondii isolates and genotyped at four ROP gene loci, including ROP5, ROP16, ROP17 and ROP18 to look for association with markers of virulence. RESULTS: Infection with ToxoDB genotype #13 from the Caribbean resulted in 100% of mice being euthanized which was comparative to infection with the virulent Brazilian strain (ToxoDB genotype #6). Significantly higher parasite burdens were recorded in the lungs and eyes of mice infected with ToxoDB genotypes #13 and #6. Genotyping of ROP loci revealed that the virulent Caribbean isolates had a different ROP18/ROP5 allelic profile (3/1) to the virulent Brazilian isolate (1/3); however, the avirulent Caribbean isolate (ToxoDB genotype #1) had the same ROP18/ROP5 profile as the avirulent European isolate (ToxoDB #3) (both 2/2). Caribbean isolates of intermediate virulence (ToxoDB #141 and #265) all had the same ROP18/ROP5 allelic profile (2/2). CONCLUSIONS: Isolates from the Caribbean with ToxoDB genotype #13 were acutely virulent for mice and comparable to a known virulent Brazilian isolate. The ROP protein allelic profile of the virulent Caribbean and Brazilian isolates differed indicating that perhaps other factors are involved in predicting virulence. Understanding virulence is important for predicting disease outcome in humans and may also aid vaccine design as well as drug discovery.

Molecular confirmation of Sarcocystis gigantea in a naturally infected sheep in Argentina: A case report.

The work describes a case of Sarcocystis gigantea infection in a 3-years-old Corriedale ewe from Buenos Aires Province, Argentina. The ewe was found dead with a poor body condition. Pathological and molecular studies were carried out in order to try and confirm the causative agent of the infection. At necropsy, approx. 100 whitish elliptic (3-5mm to 5-8mm) macrocysts with a hard consistency were observed along the esophageal and pharyngeal muscular layers. Microscopically, the macrocysts consisted of an eosinophilic wall, internal septa originated from the eosinophilic wall and basophilic parasitic cells were located among the septa. The sarcocysts were identified molecularly through PCR amplification and sequencing of a short segment of the 18S rRNA gene. Sequence analysis of the amplified DNA demonstrated 100% identity to S. gigantea sequences previously published. To our knowledge this is the first molecular confirmation of S. gigantea infection in sheep in the Americas.

Predominance of atypical genotypes of Toxoplasma gondii in free-roaming chickens in St. Kitts, West Indies.

BACKGROUND: Toxoplasma gondii is a worldwide protozoan parasite of felids which can infect almost all warm-blooded animals, including humans. Free-roaming chickens are good indicators of environmental contamination with T. gondii oocysts because they feed from the ground. Previous research has demonstrated a high seroprevalence of T. gondii in domestic animals on St. Kitts but little is known about the genotypes circulating in the environment. METHODS: Hearts and brains from 81 free-roaming chickens in St. Kitts were digested and inoculated into 243 Swiss Webster mice in a bioassay. DNA was extracted from digested chicken tissues and the brains of all mice, and screened for T. gondii. Positive samples were genotyped using restriction fragment length polymorphism. Chicken sera were also screened for T. gondii antibodies using a modified agglutination test (MAT). RESULTS: Overall, 41% (33 out of 81) of chickens were positive for T. gondii either by serology and/or by PCR. Antibodies to T. gondii were detected by MAT in 32% (26 out of 81) of chickens, and T. gondii DNA was detected in mouse brains representing 26% (21 out of 81) of chickens. Genotyping of 21 DNA isolates, using polymorphisms at 10 loci, including SAG1, SAG2 (5'-3' SAG2 and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, revealed that 7 were ToxoDB genotype #141, 6 were #1 (Type II), 3 were #13, 3 were #265, one was #264 and one was #2 (Type III). Genotypes #13 and #141 appear to be more virulent. CONCLUSIONS: The results of this study highlight the greater genetic diversity of T. gondii circulating in the Caribbean region, with potentially different degrees of virulence to humans.

Toxoplasma gondii in livestock in St. Kitts and Nevis, West Indies.

BACKGROUND: Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals including livestock. In these animals, the parasite forms cysts in the tissues which may pose a risk to public health if infected meat is consumed undercooked or raw. The aim of this study was to determine the exposure of livestock to T. gondii in St. Kitts and Nevis. METHODS: Sera and/or heart tissue and meat juice were collected from pigs (n = 124), sheep (n = 116) and goats (n = 66) at the St. Kitts Abattoir. Sera and meat juice were screened for reactive antibodies to T. gondii using an in-house ELISA. Heart tissue was screened for T. gondii DNA using quantitative PCR and positive samples were genotyped using RFLP. RESULTS: Antibodies to T. gondii were detected in sera from 48% of pigs, 26% of sheep and 34% of goats tested. Antibodies were also detected in the meat juice from 55% of pig hearts, 22% of sheep hearts and 31% of goat hearts tested. There was a significant positive correlation between serology and meat juice results. T. gondii DNA was detected in heart tissue of 21% of pigs, 16% of sheep and 23% of goats tested. Preliminary PCR-RFLP analysis identified a predominance of the Type III genotype of T. gondii. CONCLUSIONS: These results suggest widespread environmental contamination with T. gondii oocysts and that livestock could be a potentially important source of T. gondii infection if their infected meat is consumed (or handled) undercooked.

First report of Toxoplasma gondii seroprevalence in wild-caught Caribbean African green monkeys.

BACKGROUND: Toxoplasma gondii is a protozoan parasite capable of infecting all warm-blooded animals. Humans can become infected by ingesting infective oocysts from the environment or contaminated food or water, or by ingesting tissue cysts in undercooked infected meat or by handling infected meat. Caribbean African green monkeys (Chlorocebus sabaeus) are present in large numbers on the island of St. Kitts in the Caribbean, and it is not uncommon for these animals to be trapped and eaten by islanders. The aim of this study was to determine T. gondii infection in Caribbean African green monkeys. FINDINGS: Sera collected from 79 wild-caught Caribbean African green monkeys were examined for T. gondii antibodies by ELISA. Antibodies were detected in 38 out of 79 (48.1%) monkeys. Significantly more females were infected than males but there was no significant effect of age or location on antibody status. CONCLUSIONS: Results indicate that Caribbean African green monkeys can be infected with T. gondii and that there is widespread environmental contamination of St. Kitts with oocysts. These monkeys could present a potential source of T. gondii infection if their meat is consumed undercooked. This is the first report of T. gondii antibodies in this species.

Seroprevalence of Toxoplasma gondii in small ruminants from four Caribbean islands.

BACKGROUND: Toxoplasma gondii is a protozoan parasite capable of infecting all warm-blooded animals including livestock. In these animals, the parasite forms cysts in the tissues which may pose a risk to public health if infected meat is consumed undercooked or raw. Little is known of the epidemiology of T. gondii in the Caribbean; therefore, the aim of this study was to determine T. gondii exposure in small ruminants from four Caribbean island nations. FINDINGS: Sera from 305 sheep and 442 goats from Dominica, Grenada, Montserrat and St. Kitts and Nevis were examined for T. gondii antibodies using an in house ELISA. Reactive antibodies were detected in sheep and goats, respectively, from Dominica (67%, 37/55; 58%, 79/136), Grenada (48%, 40/84; 57%, 54/94), Montserrat (89%, 25/28; 80%, 25/31) and St. Kitts and Nevis (57%, 78/138; 42%, 76/181). CONCLUSIONS: Our results suggest widespread environmental contamination with T. gondii oocysts and that small ruminants could be a potentially important source of T. gondii infection if their infected meat is consumed undercooked.

Cytokine expression in the placenta of pregnant cattle after inoculation with Neospora caninum.

Neospora caninum is recognized as a major cause of reproductive losses worldwide but its pathogenesis is not completely understood. Immune mediated placental pathology has been reported as being responsible for compromising pregnancy probably due to the adverse effects of exacerbated Th1 type response at the maternal-foetal interface. Different clinical outcomes are known to occur following experimental infections of cattle at different stages of gestation, with foetal death being the most common finding during early gestation, and the birth of live congenitally infected calves following infection later in gestation. The aim of the current study was to characterize the cytokine expression in the placenta of cattle experimentally challenged with tachyzoites of the Nc-1 strain during early, mid and late gestation. Moderate to severe infiltration of IL-12, IFN-γ and TNF-α expressing cells was observed in the placentas collected at early gestation and this infiltration was more pronounced in the samples collected from challenged dams carrying non-viable foetuses, compared with the mothers carrying viable foetuses. In contrast, the infiltration of Th1 cytokine expressing-cells was mild following N. caninum infection in mid gestation and scarce during infection in late gestation. Scarce expression of IL-4 was observed in the placentas from N. caninum-challenged and negative control animals throughout gestation. The milder Th1 immune response observed during later stages of gestation following Nc-1 infection could partially explain the less severe clinical outcome when compared to early pregnancy.

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy.

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n = 8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n = 5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5 × 10(8) tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion = 12.5%), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion = 100%). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc-Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.

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.

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.

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay.

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.