Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from S?o Paulo, Brazil: unexpected findings.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding S?o Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.     

Toxoplasma gondii isolates from free-ranging chickens from the United States

Prevalence of Toxoplasma gondii infection in chickens is a good indicator of the strains prevalent in their environment because they feed from ground. The prevalence of T. gondii was determined in 118 free-range chickens from 14 counties in Ohio and in 11 chickens from a pig farm in Massachusetts. Toxoplasma gondii antibodies (> or = 1: 5) were found using the modified agglutination test (MAT) in 20 of 118 chickens from Ohio. Viable T. gondii was recovered from 11 of 20 seropositive chickens by bioassay of their hearts and brains into mice. The parasite was not isolated from tissues of 63 seronegative (< or = 1:5) chickens by bioassay in cats. Hearts, brains, and muscles from legs and breast of the 11 chickens from the pig farm in Massachusetts were fed each to a T. gondii-negative cat. Eight cats fed chicken tissues shed oocysts; the 3 cats that did not shed oocysts were fed tissues of chickens with MAT titers of 1:5 or less. Tachyzoites of 19 isolates of T. gondii from Ohio and Massachusetts were considered avirulent for mice. Of 19 isolates genotyped, 5 isolates were type II and 14 were type III; mixed types and type I isolates were not found.     

Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies

Cats are important in the natural epidemiology of Toxoplasma gondii, because they are the only hosts that can excrete environmentally resistant oocysts. Cats are infected with T. gondii via predation on infected birds and rodents. During 2005, 238 rats (Rattus norvegicus) were trapped in Grenada, West Indies, and their sera along with tissue samples from their hearts and brains were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test (MAT, titer 1:40 or higher); only 2 (0.8%) of 238 rats were found to be infected. Brains and hearts of all rats were bioassayed in mice. Toxoplasma gondii was isolated from the brain and the heart of only 1 rat, which had a MAT titer of 1:320. All of 5 mice inoculated with the heart tissue, and the 5 mice inoculated with the brain tissue of the infected rat remained asymptomatic, even though tissue cysts were found in their brains. Genetically, the isolates of T. gondii from the heart and the brain were identical and had genotype III by using the SAG1, SAG2, SAG3, BTUB, and GRA6 gene markers. These data indicate that rats are not important in the natural history of T. gondii in Grenada.     

Cellular responses of cats with primary toxoplasmosis.

The cellular responses of 8 kittens (4 inoculated orally with mouse brains containing Toxoplasma gondii cysts and 4 uninfected controls) were studied. Oocyst numbers, body weight, and rectal temperature were monitored daily. Blood was collected weekly for serology and mononuclear cell purification. At necropsy, peritoneal and alveolar macrophages, spleen, and lymph node cells were harvested. Infected cats shed oocysts 4-15 days postinfection, maintained normal body weight and rectal temperature, and developed anti-T. gondii immunoglobulin M and G. Infected cats had normal surface immunoglobulin-positive cell populations and peripheral blood lymphocyte functions. The infected cats differed in their responses from control cats in that they developed circulating T. gondii antigen-specific lymphocytes, had increased interleukin 1 secretion by monocytes, had spleen and lymph node cells with depressed mitogenic responses and interleukin 2 production, and had macrophages with enhanced abilities in preventing the intracellular proliferation of T. gondii. Overall, the primary response of the cat to an infection with T. gondii appeared similar to that of other hosts.     

Toxoplasma gondii infections in cats from Paraná, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. The prevalence of T. gondii was determined in 58 domestic cats from 51 homes from Santa Isabel do Ivai, Parana State, Brazil where a water-associated outbreak of acute toxoplasmosis had occurred in humans. Antibodies to T. gondii were found with the modified agglutination test in 49 of 58 (84.4%) cats at a serum dilution of 1:20. Tissues (brain, heart, and skeletal muscle) of 54 of these cats were bioassayed in T. gondii-free, laboratory-reared cats; T. gondii oocysts were excreted by 33 cats that were fed feline tissues. Brains from these 54 cats were bioassayed in mice; T. gondii was isolated from 7. Skeletal muscles and hearts of 15 cats were also bioassayed in mice; T. gondii was isolated from skeletal muscles of 9 and hearts of 13. The results indicate that T. gondii localizes in muscle tissue more than the brains of cats. In total there were 37 T. gondii isolates from 54 cats. Most isolates of T. gondii were virulent for mice. Genotyping of the 37 isolates of T. gondii, using the SAG2 locus, revealed that 15 isolates were type I and 22 were type III. The absence of type II genotype in cats in this study is consistent with the previous studies on T. gondii isolates from Brazil and is noteworthy because most T. gondii isolates from the United States are type II. These findings support the view that Brazilian and North American T. gondii isolates are genetically distinct. This is the first report of genotyping of T. gondii isolates from the domestic cat.     

Oocyst shedding by cats fed isolated bradyzoites and comparison of infectivity of bradyzoites of the VEG strain Toxoplasma gondii to cats and mice

Infectivity of bradyzoites of the VEG strain of Toxoplasma gondii was compared in cats and mice. For this, tissue cysts were separated from brains of infected mice using a Percoll gradient, and bradyzoites were released by incubation in acidic pepsin solution. After filtration through a 3-microm filter, bradyzoites were counted and diluted 10-fold in RPMI tissue culture medium. Dilutions estimated to have 1, 10, 100, and 1,000 bradyzoites were fed to cats and inoculated into mice, orally or subcutaneously (s.c.). Three experiments were performed. In experiment 1, 2 of 2 cats fed 1,000 bradyzoites, 1 of 2 cats fed 100 bradyzoites, 1 of 4 cats fed 10 bradyzoites, and 1 of 4 cats fed 1 bradyzoite shed millions of oocysts; 1,000 bradyzoites were infective to all 4 inoculated mice s.c. but not to 4 mice inoculated orally, and 100 bradyzoites were infective to 2 of 4 mice injected s.c. but not to 4 mice inoculated orally. All 16 mice (8 oral, 8 s.c.) injected with 1 or 10 bradyzoites were negative for T. gondii. In experiment 2, 1 of 4 cats fed 10 counted bradyzoites shed oocysts; the same inocula were not infective to 4 mice injected s.c. In experiment 3, 3 of 4 cats fed 1,000 bradyzoites shed oocysts and the inocula were infective to 10 of 10 mice s.c. and 4 of 10 mice orally; 4 of 4 cats fed 100 bradyzoites shed oocysts and the inocula were infective to 6 of 10 mice s.c. and 0 of 10 mice orally; 10 bradyzoites were not infective to cats and mice. Results indicate that bradyzoites are more infective to cats than to mice, and cats can shed millions of oocysts after ingesting just a few bradyzoites.     

Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase

The role of B cells in resistance against Toxoplasma gondii was studied using B cell-deficient (muMT) mice. Following peroral infection with 10 cysts of the ME49 strain, all muMT mice survived the acute stage of the infection but died between 3 and 4 wk after infection. In contrast, all control mice were alive at 8 wk after infection. At the stage during which muMT animals succumbed to the infection, parasite replication and pathology were most evident in their brains; small numbers of tachyzoites were also detectable in their lungs. Significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites were observed in brains of muMT than in control mice. Large areas of necrosis associated with numerous tachyzoites were observed only in brains of muMT mice. Anti-T. gondii IgG Abs were detected only in sera of control mice, whereas similar levels of IFN-gamma were detected in sera of both strains of mice. Amounts of mRNA for IFN-gamma, IL-10, and inducible NO synthase in the brain did not differ between infected muMT and control mice. Expression of mRNA for TNF-alpha was increased in brains of muMT mice. Administration of polyclonal rabbit anti-T. gondii IgG Ab prevented early mortality and pathology associated with tachyzoites in the brain in the infected muMT mice. These results indicate that B cells play an important role, most likely through their production of specific Abs, in resistance to persistent active (tachyzoite) infection with T. gondii in mice, especially in the brain and lung.     

Tissue distribution and molecular characterization of chicken isolates of Toxoplasma gondii from Peru

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii antibodies in sera of 50 free-range chickens (Gallus domesticus) from Peru was 26% on the basis of the modified agglutination test (MAT). Hearts, pectoral muscles, and brains of seropositive (MAT > or =1:5) chickens were bioassayed individually in mice. Tissues from the remaining 37 seronegative chickens were pooled and fed to 2 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. Toxoplasma gondii was isolated from the hearts of 10 seropositive chickens but not from their brains and pectoral muscles. Genotyping of these isolates using the SAG2 locus indicated that 7 isolates were type I and 3 were type III. Six of the 7 type-I isolates were avirulent for mice, which was unusual because type-I isolates are considered virulent for mice. The T. gondii isolates were from chickens from different properties that were at least 200 m apart. Thus, each isolate is likely to be different. This is the first report of isolation of T. gondii from chickens from Peru.     

Low virulence of oocysts of Czech Toxoplasma gondii isolates on the basis of biological and genetic characteristics

The virulence of the oocysts of 7 Czech Toxoplasma gondii isolates was tested. The oocysts were obtained by experimental infection of cats with the tissue cysts of T. gondii isolates from dogs, cats, and rabbits. The cats shed the oocysts in feces, with prepatent periods of 3-5 days postinfection (PI); the patent period was 7-18 days. The number of oocysts shed varied between 0.94 million and 47 million, with 0.66 million-39 million oocysts found in the daily samples of excrement. The cats ceased oocyst production at 11-22 days PI. Sporulated oocysts were used to prepare infective doses of 1 to 10(5) oocysts for oral infection of 10 mice. Deoxyribonucleic acid isolated from 4 T. gondii isolates was used in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for amplification of the ROP1 gene and restriction of the product of amplification by restriction endonuclease DdeI. On the basis of their biological characteristics, all 7 isolates belonged to the group of "avirulent" strains. In the PCR-RFLP tests, 2 isolates, K9 and K19, showed an "avirulent" strain pattern.     

High prevalence of toxoplasmosis in cats from Egypt: isolation of viable Toxoplasma gondii, tissue distribution, and isolate designation

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, 158 feral cats from Giza, Egypt, were examined for T. gondii infection. Antibodies to T. gondii were found in 97.4% with the modified agglutination test. Viable T. gondii was isolated from tissues (brain, heart, tongue) of 115 of 137 cats by bioassay in mice. These isolates were designated TgCatEg 1-115; none of these isolates was virulent to out-bred Swiss Webster mice. Of the 112 seropositive cats whose tissues were bioassayed individually, T. gondii was isolated from the hearts of 83 (74.1%), tongues of 53 (47.3%), and brains of 36 (32.1%). Toxoplasma gondii oocysts were not detected in rectal contents of any of the 158 cats, probably related to high seropositivity (chronic infection) of cats surveyed. The high prevalence of T. gondii in feral cats in Egypt reported here indicates a high environmental contamination with oocysts.     

A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine

A 3-yr field trial was conducted on 8 commercial swine farms in Illinois to determine the effectiveness of a feline Toxoplasma gondii vaccine in reducing the exposure of swine to T. gondii. A vaccine consisting of live bradyzoites of the mutant T-263 strain, capable of preventing oocyst shedding by cats, was used in this study. Each farm was visited 3 times in 1994, 3 times in 1995, and once in 1996. Cats were trapped and inoculated with the T-263 oral vaccine during 1994 and 1995. On each visit, the following samples were collected: blood from pigs, cats, and mice for detection of serum antibodies to T. gondii, feces from cats to detect oocysts, and heart and brain tissues from rodents to determine the presence of T. gondii tissue cysts. The modified agglutination test (MAT), with a positive titer set at the 1:25 dilution, was used to determine serum antibodies. At first capture, 72.6% (61/84) of juvenile cats and 32.6% (31/95) of adult cats had no detectable antibodies (seronegative), indicating no prior exposure to T. gondii when they received their first vaccine. Of these first-time seronegative cats, 58.1% (18/31) of adult and 45.9% (28/61) of juvenile cats were recaptured and received a second dose of vaccine. Changes in the prevalence of T. gondii infection were evaluated from the prevaccination (1992, 1993) to the postvaccination (1996) period. Eleven cats (5%) were detected shedding oocysts between 1994 and 1996, of which 10 (90.1%) shed during 1994. The last detection of oocyst shedding by cats was during the first farm visit in 1995. There was a significant decrease in T. gondii seroprevalence for finishing pigs (P < 0.05, Wilcoxon sign rank test). There was a positive correlation (Spearman's p = 1.0, P < 0.0001) between the change in prevalence in juvenile cats and the change in prevalence in finishing pigs. The seropositivity rate (MAT > or = 1:25) in mice among all farms decreased from 4% in 1992-1993 to 0% in 1996. The mean prevalence of T. gondii tissue cyst isolation for mice on all farms decreased from 1.1% in 1994, to 0.8% in 1995, and to 0.5% in 1996. The results of this study suggest that the reduced exposure of pigs to T. gondii was due to the administration of the T. gondii vaccine to cats.     

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from chickens in Grenada, West Indies

The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.     

Isolation of Toxoplasma gondii from the keel-billed toucan (Ramphastos sulfuratus) from Costa Rica

Pectoral muscles from a captive keel-billed toucan (Ramphastos sulfuratus) from Costa Rica were fed to a Toxoplasma gondii-free cat, and the cat shed oocysts. Laboratory mice fed these oocysts developed antibodies to T. gondii in their sera and T. gondii tissue cysts in their brains. The DNA extracted from the brains of infected mice was characterized using 10 polymerase chain reaction-restricted fragment length polymorphic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The isolate designated TgRsCrl was found to be non-clonal with Type I, II, and III alleles at different loci. This is the first isolation of T. gondii from this host.     

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue is difficult because the number of latent cysts is small and reactivation is a transient event. To detect the early stage of reactivation in mouse brain tissue, we constructed a cyst-forming strain of T. gondii in the tachyzoite stage, specifically expressing red fluorescence. The PLK strain of T. gondii was stably transfected with a red fluorescent protein gene, DsRed Express, under the control of a tachyzoite-specific SAG-1 promoter and the resulting parasite was designated as PLK/RED. Tachyzoites of PLK/RED growing in Vero cells showed red fluorescence. When C57BL/6J mice were i.p. infected with tachyzoites of PLK/RED, red fluorescent tachyzoites were detected in their brains at the fourth day p.i. However, red fluorescent tachyzoites were not detected in BALB/c mice latently infected with PLK/RED, although non-fluorescent cysts were detected in their brains. After treatment of latently infected mice with dexamethasone for 1 month, the mice showed neurological symptoms. In mice with symptoms, red fluorescent tachyzoites were again detected in their brains and in other organs. To detect the initial site of reactivation, BALB/c mice latently infected with the strain were treated with dexamethasone for 3 weeks, and brains were excised before any symptoms appeared. Excised brains were examined for red fluorescence-positive sites. By a histological study of red fluorescent-positive sites, we detected a cyst containing red fluorescent zoites, which still had a PAS stain-positive cyst wall. A few red fluorescent zoites breaking away from the cyst were also observed. The stage-specific expression of fluorescent protein facilitates detection of a rare transient event and makes it possible to detect the initial site of reactivation.     

Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR

We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.     

Characterization of Toxoplasma gondii isolates in free-range chickens from Amazon, Brazil

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 50 free-range chickens (Gallus domesticus) from Amazon, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 33 (66%) chickens with titers of 1:5 in 3, 1:10 in 2, 1:20 in 1, 1:40 in 1, 1:80 in 2, 1:160 in 5, 1:200 in 9, 1:400 in 5, 1:800 in 2, 1:1,600 in 2, and 1:3,200 or higher in 1. Hearts and brains of 33 seropositive chickens were bioassayed individually in mice. Tissues from 17 seronegative chickens were pooled and fed to 2 T. gondii-free cats. Feces of cats were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 24 chickens with MAT titers of 1:5 or higher. Genotyping of these 24 T. gondii isolates by polymorphisms at the SAG2 locus indicated that 14 were type I, and 10 were type III; the absence of type II strains from Brazil was confirmed. Fifty percent of the infected mice died of toxoplasmosis, irrespective of the genotype.     

Effect of high temperature on infectivity of Toxoplasma gondii tissue cysts in pork

To study the effect of high temperature on infectivity of Toxoplasma gondii tissue cysts, pork from infected pigs was mixed with infected mouse brains and homogenized thoroughly. Twenty-gram samples of infected homogenized meat were sealed in plastic pouches, pressed to a uniform thickness of 2 mm, and subjected to water-bath temperatures of 49, 52, 55, 58, 61, 64, and 67 C for 0.01, 3, 6, 9, 12, 24, 48, and 96 min. Treated samples were digested in HCl-pepsin solution and bioassayed in mice. Toxoplasma gondii tissue cysts remained viable at 52 C for 9.5 min but not for 9.5 min at 58 C; tissue cysts were generally rendered nonviable by heating to 61 C or higher temperature for 3.6 min. Tissue cysts survived once at 64 C for 3 min. These data demonstrate that T. gondii tissue cysts are less heat resistant than encysted Trichinella spiralis larvae.     

Diverse and atypical genotypes identified in Toxoplasma gondii from dogs in São Paulo, Brazil

The prevalence of Toxoplasma gondii in 118 unwanted dogs from S?o Paulo City, S?o Paulo State, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test and found in 42 (35.8%) dogs, with titers of 1:20 in 10, 1:40 in 6, 1:80 in 5, 1:160 in 5, 1:320 in 6, 1:640 in 7, and 1:1,280 or higher in 3. Hearts and brains of 36 seropositive dogs were bioassayed in mice, or cats, or both. Tissues from 20 seropositive dogs were fed to 20 T. gondii-free cats. Feces of cats were examined for oocysts. Toxoplasma gondii was isolated from 15 dogs by a bioassay in mice, from the brain alone of 1, from the heart alone of 4, and from both brains and hearts of 10. All infected mice from 5 of 15 isolates died of toxoplasmosis during primary infection. Four additional isolates were obtained by bioassay in cats. Genotyping of these 19 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 (an apicoplast marker Apico) revealed 12 genotypes. One isolate had Type III alleles at all 11 loci, and the remaining 18 isolates contained a combination of different alleles and were divided into 11 genotypes. The absence of Type II in Brazil was confirmed. The result supports previous findings that T. gondii population genetics is highly diverse in Brazil.     

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.     

Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites

Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures. Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined. Tissue cysts were first seen 3 days postinoculation (PI) using TEM. Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation. Cats fed cell cultures excreted T. gondii oocysts in their feces 5-7 days PI. These oocysts caused lethal infections in mice. Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites. Tissue cyst formation has been followed through 40 subpassages of infected cells. By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted. This indicates that the parasite had become oocystless after repeated passage in vitro.