Oocyst-induced Toxoplasma gondii infections in cats

To investigate the oocyst-induced cycle with a 21+ day prepatent period, 32 cats were fed 5 x 10(5) to 2 x 10(7) sporulated oocysts of Toxoplasma gondii and necropsied between 4 hr and 41 days thereafter. The presence of the earliest stages in 7 cats was tested in mice. The tissues of 25 cats were studied histologically; 17 were bioassayed by feeding them to cats to determine, by the length of the prepatent period, whether bradyzoites were present. Based on previous studies, a short (3-10 days) prepatent period indicated that bradyzoites were present in an oral inoculum and a long (greater than 21 days) prepatent period indicated the presence of tachyzoites only. Tissues from 14 cats were also bioassayed in mice for the presence of bradyzoites, using their resistance to pepsin as indicator. Six were studied by both methods. Based on these criteria, tachyzoites predominated in extraintestinal organs during the first 14 days after infection. They were found as early as 4 hr in mesenteric lymph nodes where their number reached 10(4) after 6 and 9 days; they were present after 1 day in all levels of the small intestine and after 6 days in the liver, lung, and blood. Bradyzoites were first detected 10 days after oocyst feeding; they predominated by the third week of infection and were present up to 41 days. Enteroepithelial stages were found histologically only in 2 cats, 24 and 41 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lytic cycle of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. This protozoan parasite is one of the most widespread, with a broad host range including many birds and mammals and a geographic range that is nearly worldwide. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immunocompromised. This sophisticated eukaryote has many specialized features that make it well suited to its intracellular lifestyle. In this review, we describe the current knowledge of how the asexual tachyzoite stage of Toxoplasma attaches to, invades, replicates in, and exits the host cell. Since this process is closely analogous to the way in which viruses reproduce, we refer to it as the Toxoplasma "lytic cycle."     

Detection of Toxoplasma gondii parasitemia in experimentally inoculated cats

Toxoplasma gondii B1 gene polymerase chain reaction (PCR) amplification utilizing a flanking and nesting reaction was compared to mouse bioassay on feline whole blood samples collected before and after experimental inoculation with T. gondii. Samples were collected from 5 cats prior to inoculation with T. gondii and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 112, 140, 143, 147, 150, 154, 161, 168, 175, and 182 after inoculation. Cats were challenged with T. gondii orally on day 140. Bioassay was found to be less effective for detection of parasitemia than B1 gene PCR. Parasitemia was detected in all 5 cats by PCR multiple times after primary and challenge inoculation. Detection of T. gondii parasitemia by PCR utilizing the flanking reaction described here may be useful in predicting the oocyst shedding period in individual cats. As none of the cats developed signs of systemic illness, yet were chronically parasitemic, T. gondii whole-blood PCR is not helpful as a diagnostic test for clinical feline toxoplasmosis.     

Molecular characterization of Toxoplasma gondii isolates from cats in Spain

Cats are important in the epidemiology of Toxoplasma gondii because felids are the only definitive hosts that can excrete environmentally resistant oocysts. Fresh samples of brain from 103 Spanish cats with antibodies to T. gondii were analyzed for T. gondii DNA using nested-PCR; 47 (45.5%) were found to be positive. Further characterization of DNA from 46 cats using RFLP-PCR at the 3' and 5' ends of the SAG2 locus revealed that 12 (26%) isolates were Type I and 34 (74%) were Type II; no Type III were found, and the 47th sample could not be classified to its genetic type. In addition, T. gondii was also isolated by bioassay in mice from 42 of 103 seropositive cats. This is the first report of T. gondii characterization from cats in Spain.     

Prevalence of Toxoplasma gondii in stray cats of Gyeonggi-do, Korea

Toxoplasma gondii is an obligate intracellular zoonotic protozoan with a worldwide distribution. It infects humans as well as a broad spectrum of vertebrate hosts. Cats and wild felidae play crucial roles in the epidemiology of toxoplasmosis. This study was performed to survey the prevalence of T. gondii infection among stray cats in the Gyeonggi-do, Republic of Korea. A total of 174 stray cat blood samples were collected from Gwacheon-si (n=20), Bucheon-si (82), and Yangju-si (72). Positive sera for T. gondii were identified in 14 samples (8.1%) exclusively via the latex agglutination test, 28 (16.1%) via ELISA, and 23 (13.2%) via PCR analysis. The overall infection rate of female stray cats (29.2%) presented as higher than that of male cats (24.0%). This study suggests that T. gondii is widespread in the stray cat population of Gyeonggi-do, Korea. It is urgently needed to control urban stray cat population and to reduce the risk of zoonotic transmission of toxoplasmosis to other animal hosts and humans.     

Prevalence of Toxoplasma gondii antibodies in domestic cats from rural Ohio

Antibodies to Toxoplasma gondii were determined in serum samples from 275 domestic cats from a mobile spay and neuter clinic from 8 counties in Ohio. The modified agglutination test incorporating whole formalinized tachyzoites and mercaptoethanol was used to determine antibodies. Antibodies to T. gondii were found in 133 (48%) out of 275 cats: in titers of 1:25 in 24, 1:50 in 37, and 1:500 or more in 72. The highest prevalence (62% of 78) was in outdoor cats. The prevalence of T. gondii antibodies in 48% of cats suggests wide-spread contamination of the rural environment with oocysts.     

Seroprevalence of Toxoplasma gondii in cats from Colombo, Sri Lanka

Cats are essential in the life cycle of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Nothing is known of the prevalence of Toxoplasma gondii in cats from Sri Lanka. Serum samples from 86 cats from Colombo, Sri Lanka, were tested for antibodies to T. gondii using the modified agglutination test; antibodies were found in 26 (30.2%) cats with titers of 1:25 in 4, 1:50 in 4, 1:100 in 3, 1:400 in 2, 1:800 in 3, 1:1,600 in 4, and 1:3,200 or higher in 6 cats. Seropositivity increased with age and was higher in stray cats versus pet cats. This is the first report of seroprevalence of T. gondii in cats from Sri Lanka.     

Toxoplasma gondii: cross-immunity against the enteric cycle

Eight of nine cats inoculated with strain ME-49 and challenged with three different strains of Toxoplasma were immune to oocyst shedding, as ascertained with bioassays of their feces. In a second experiment, only toxoplasma asexual stages were seen in H&E stained gut sections of cats treated with suppressive doses of sulfamerazine and pyrimethamine starting 2 days after oral inoculation with cysts of the strain ME-49 and killed 6 days later. In a third experiment, four cats were similarly inoculated and treated for 20 days. Six weeks later, the cats received an oral homologous challenge with cysts, and none shed toxoplasma oocysts. An acceptable level of cross-protection was achieved with strain ME-49, and therefore, it can be used as a candidate strain from which antigens could be tested for enteric protection.     

Toxoplasma gondii seroprevalence in goats,cats and humans in Russia

Toxoplasmosis, a most common zoonosis, is caused by the protozoan parasite Toxoplasma gondii. However, there is little epidemiological information on T. gondii infections in humans and livestock animals in Russia. Therefore, in this study, the seroprevalence of T. gondii in goats in Russia was investigated. A total of 216 goats from 32 farms were investigated and 95 of them were seropositive for T. gondii. The difference in seroprevalence between the examined regions was not statistically significant. We next collected serum samples from 99 cats and 181 humans in Kazan city, the state capital of the Republic of Tatarstan, Russia, and examined their T. gondii seroprevalences. Thirty-nine of the 99 cat samples and 56 of the 181 human samples showed seropositivity. Logistical regression analysis revealed that the cat breeding history of the human subjects, but not their sex or age is a significant risk factor for T. gondii seropositivity. These findings suggest that the natural environment in Russia may be widely polluted with T. gondii oocysts shed by cats, and ingestion of these oocysts provides a major route for human infection with this parasite.     

Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts

Two Toxoplasma gondii genes were characterized that are differentially expressed during the parasite's life cycle. The genes named LDH1 and LDH2, respectively, encode polypeptides similar to the enzyme lactate dehydrogenase (LDH; l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27) from a variety of organisms. They show 64.0% nucleotide identity in the coding region and both have an intron at the same relative position. The deduced amino acid sequences of LDH1 and LDH2 share 71.1% identity. LDH1 and LDH2 are most similar to an LDH of Plasmodium falciparum (46.5% and 48.5% amino acid identities, respectively). The mRNA of LDH2 was only detected in the bradyzoite stage, while the mRNA of LDH1 was detected in both the bradyzoite and tachyzoite stages. However, by isoelectric focusing and immunoblot analysis, only one LDH isoform was found to be expressed in each stage. Furthermore, the expression of a reporter gene carrying chloramphenicol acetyltransferase (CAT) coding sequence and the putative LDH2 promoter sequence was significantly up-regulated by growing parasites in tissue culture in media with alkaline pH (pH 8.2, a condition known to induce the expression of bradyzoite-specific antigens), while the expression of a CAT reporter construct carrying the putative LDH1 promoter sequence was down-regulated by similar treatment. These results indicate that LDH expression is developmentally regulated in T. gondii and suggest a possible correlation between stage conversion and alteration in carbohydrate or energy metabolism in this parasite.     

Prevalence of Toxoplasma gondii infection in feral cats in Seoul, Korea

The present study assessed the prevalence of Toxoplasma gondii infection in feral cat populations in Seoul using enzyme-linked immunosorbent assay (ELISA) and nested polymerase chain reaction (PCR). A total of 456 feral cats from 17 wards in Seoul was surveyed. The overall prevalence of T. gondii infection was 15.8% (69/456) by ELISA and 17.5% (80/456) by PCR; by gender, 17% (44/259) by ELISA and 16.2% (42/259) by PCR in males and 14.3% (28/196) by ELISA and 19.4% (38/196) by PCR in females. On a baseline of the Han River, prevalence was 15.1% (29/192) by ELISA and 15.6% (30/192) by PCR in the upper region and 16.4% (43/264) by ELISA and 18.9% (50/264) by PCR in the lower area. This suggested that toxoplasmosis is widespread throughout Seoul's feral cat population and it is critical that the city institute policies for the control of the feral cat population to reduce the risk of toxoplasmosis transmission to animals, including humans.     

Toxoplasma gondii: perfecting an intracellular life style

Toxoplasma gondii is a widespread protozoan parasite that infects all nucleated cell types of warm-blooded vertebrates. Parasite motility is regulated by polymerization of new actin filaments that provide a substrate for the small myosin TgMyoA. Interaction between the cytoplasmic tails of parasite adhesins and the actin-binding protein aldolase links these cell surface proteins with the cytoskeleton. Translocation of adhesins coupled to extracellular receptors allows the parasite to glide across the substrate. This conserved system is important for active penetration into host cells and tissue migration by T. gondii . Entry into the host cell is accompanied by dramatic remodeling of the intracellular vacuole that the parasite resides in. This compartment resists fusion with host cell endocytic organelles, yet recruits mitochondria and endoplasmic reticulum in order to gain access to host cell nutrients. The combined abilities to actively penetrate host cells and control the fate of the parasite-containing vacuole contributes to the remarkable success of T. gondii as an intracellular parasite.     

Seroprevalence of Toxoplasma gondii antibodies in cats from Durango City, Mexico

The prevalence of antibodies to Toxoplasma gondii was determined in sera from 105 domestic cats from Durango City, Mexico. Using a modified agglutination test, antibodies to this parasite were found in 21% of the 105 cats, with titers of 1:25 in 3 cats, 1:50 in 4 cats, 1:200 in 5 cats, 1:400 in 2 cats, 1:800 in 2 cats, 1:1,600 in 4 cats, and 1:3,200 or higher in 2 cats. Cats older than 1 yr had a significantly higher frequency of infection than that found in cats younger than 0.5 yr (41 vs. 13.2%, respectively; odds ratio = 4.55; 95% CI = 1.24-17.18; P = 0.01). Overall, the seroprevalence of T. gondii antibodies in cats in Durango, Mexico, is much lower compared with those reported in other countries.     

Seroprevalence of Toxoplasma gondii antibodies in domestic cats from Barcelona, Spain

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts. Antibodies to T. gondii were determined in serum samples from 220 domestic cats (Felis catus) from Barcelona, Spain, using the modified agglutination test (MAT). Antibodies to T. gondii were found in 99 (45%) of 220 cats, with MAT titers of 1:25 in 26, 1:50 in 57, and > or = 1:500 in 16 cats. Seropositivity (MAT 1:25 or more) was significantly higher in adult (> or = 1 yr old, 49.7% of 153) than in juvenile (< 1 yr old, 34.3% of 67) cats, in feral (51.9% of 131) than in domiciled (34.8% of 89) cats, and in cats living in a group (community) of more than 5 cats (50.7% of 142) than in cats living alone (28.0% of 50). These seropositive cats are likely to have already shed T. gondii oocysts in the environment around Barcelona.     

Toxoplasma gondii: characterization of temperature-sensitive tachyzoite cell cycle mutants

We mutagenized RH delta hxgprt strain tachyzoites of Toxoplasma gondii using N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutants) that were unable to grow in cell culture at 40 degrees C. These isolates grew normally at 34 degrees C, but showed variable growth at temperatures between 34 and 39 degrees C. The inability to grow at 40 degrees C was also correlated with a loss of virulence in mice for those mutants examined. We further characterized the temperature-sensitive (ts) isolates using flow cytometry and propidium iodide staining and identified three types of cell cycle-related mutations. Regardless of temperature, in the isolates ts1C12, ts7B4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (congruent with 85%) than that observed for RH delta hxgprt (congruent with 60%). Four other isolates, ts4F6, ts6C11, ts8G10, and ts11F5, contained G1-related mutations, and in each case, the DNA distribution among parasites at the permissive temperature was similar to that of the parental strain, but at 40 degrees C only a single population containing a 1N nuclear DNA complement was evident. Furthermore, there was no evidence of nuclear division or cytokinesis at 40 degrees C, and these parasites demonstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populations with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 parasites contained a single nucleus, and a major subfraction of the 2N population contained abnormal and incompletely formed daughters-indicating that the initiation of daughter formation can occur in the absence of nuclear division.     

Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant

Growth rate is a major pathogenesis factor in the parasite Toxoplasma gondii; however, how cell division is controlled in this protozoan is poorly understood. Herein, we show that centrosomal duplication is an indicator of S phase entry while centrosome migration marks mitotic entry. Using the pattern of centrosomal replication, we confirmed that mutant ts11C9 undergoes a bimodal cell cycle arrest that is characterized by two subpopulations containing either single or duplicated centrosomes which correlate with the bipartite genome distribution observed at the non-permissive temperature. Genetic rescue of ts11C9 was performed using a parental RH strain cDNA library, and the cDNA responsible for conferring temperature resistance (growth at 40 degrees C) was recovered by recombination cloning. A single T. gondii gene encoding the protein homologue of XPMC2 was responsible for genetic rescue of the temperature-sensitive defect in ts11C9 parasites. This protein is a known suppressor of mitotic defects, and in tachyzoites, TgXPMC2-YFP localized to the parasite nucleus and nucleolus which is consistent with the expected subcellular localization of critical mitotic factors. Altogether, these results demonstrate that ts11C9 is a conditional mitotic mutant containing a single defect which influences two distinct control points in the T. gondii tachyzoite cell cycle.