Dolores E. Hill

Biology and epidemiology of Toxoplasma gondii in man and animals.

Abstract Toxoplasma gondii is a coccidian parasite which utilizes felids as definitive hosts, and which has an unusually wide intermediate host range. The parasite was initially described by Nicolle and Manceaux in 1908 from the rodent, Ctenodactylus gundi. Infection with T. gondii is one of the most common parasitic infections of man and other warm-blooded animals. It has been found worldwide from Alaska to Australia. Nearly one-third of humanity has been exposed to this parasite; serologic surveys indicate that T. gondii infections are common in wild carnivores, including pigs, bears, felids, fox, raccoons, and skunks. Clinical and subclinical toxoplasmosis has been reported from wild cervids, ungulates, marsupials, monkeys, and marine mammals. Southern sea otter populations have been severely impacted by Toxoplasma infections.

Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from São 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 São 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.

PREVALENCE OF VIABLE TOXOPLASMA GONDII IN BEEF, CHICKEN, AND PORK FROM RETAIL MEAT STORES IN THE UNITED STATES: RISK ASSESSMENT TO CONSUMERS

The prevalence of viable Toxoplasma gondii was determined in 6,282 samples (2,094 each of beef, chicken, and pork) obtained from 698 retail meat stores from 28 major geographic areas of the United States. Each sample consisted of a minimum of 1 kg of meat purchased from the retail meat case. To detect viable T. gondii, meat samples were fed to T. gondii-free cats and feces of cats were examined for oocyst shedding. Initially, 100 g of meat from 6 individual samples of a given species were pooled (total, 600 g), fed to a cat over a period of 3 days, and feces were examined for oocysts for 14 days; the remaining meat samples were stored at 4 C for 14 days (until results of the initial cat fecal examination were known). When a cat fed pooled samples had shed oocysts, 6 individual meat samples from each pool were bioassayed for T. gondii in cats and mice. Toxoplasma gondii isolates were then genetically characterized using the SAG2 locus and 5 hypervariable microsatellite loci. In all, 7 cats fed pooled pork samples shed oocysts. Toxoplasma gondii oocysts were detected microscopically in the feces of 2 of the cats; 1 isolate was Type II and the second was Type III. Analyzed individually, T. gondii was detected by bioassay in 3 of the 12 associated samples with genetic data indicating T. gondii isolates present in 2. The remaining 5 pooled pork samples had so few oocysts that they were not initially detected by microscopic examination, but rather by mouse bioassay of cat feces. Two were Type I, 1 was Type II, and 2 were Type III. None of the cats fed chicken or beef samples shed oocysts. Overall, the prevalence of viable T. gondii in retail meat was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat, and in particular, pork. Cooking meat to an internal temperature of 66 C kills T. gondii.

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

First isolation of Neospora caninum from the feces of a naturally infected dog.

Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum agglutination test, and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma-interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from fecal oocysts of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and polymerase chain reaction demonstrates that the dog is a natural definitive host for N. caninum.

High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA.

Little information is available on the presence of viable Toxoplasma gondii in tissues of lambs worldwide. The prevalence of T. gondii was determined in 383 lambs (<1 year old) from Maryland, Virginia and West Virginia, USA. Hearts of 383 lambs were obtained from a slaughter house on the day of killing. Blood removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Sera were first screened using 1:25, 1:50, 1: 100 and 1:200 dilutions, and hearts were selected for bioassay for T. gondii. Antibodies (MAT, 1:25 or higher) to T. gondii were found in 104 (27.1%) of 383 lambs. Hearts of 68 seropositive lambs were used for isolation of viable T. gondii by bioassay in cats, mice or both. For bioassays in cats, the entire myocardium or 500g was chopped and fed to cats, one cat per heart and faeces of the recipient cats were examined for shedding of T. gondii oocysts. For bioassays in mice, 50g of the myocardium was digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. In total, 53 isolates of T. gondii were obtained from 68 seropositive lambs. Genotyping of the 53 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed 57 strains with 15 genotypes. Four lambs had infections with two T. gondii genotypes. Twenty-six (45.6%) strains belong to the clonal Type II lineage (these strains can be further divided into two groups based on alleles at locus Apico). Eight (15.7%) strains belong to the Type III lineage. The remaining 22 strains were divided into 11 atypical genotypes. These results indicate high parasite prevalence and high genetic diversity of T. gondii in lambs, which has important implications in public health. We believe this is the first in-depth genetic analysis of T. gondii isolates from sheep in the USA.

HIGH PREVALENCE OF VIABLE TOXOPLASMA GONDII INFECTION IN MARKET WEIGHT PIGS FROM A FARM IN MASSACHUSETTS

The ingestion of uncooked infected meat is considered important in the epidemiology of Toxoplasma gondii infection in humans and little is known of the prevalence of viable T. gondii in meat used for human consumption in the United States. In the present study, viable T. gondii was isolated from 51 out of 55 pigs destined for human consumption. Hearts and tongues (500 g) from fifty-five 6-mo-old pigs from a farm in Massachusetts were bioassayed for T. gondii by feeding them to T. gondii–free cats. Feces of these cats were examined for shedding of T. gondii oocysts. Fifty-one of 55 cats fed pig tissues each shed 25–810 million T. gondii oocysts in their feces. Two of these cats consumed tissues of pigs that were shown to be seronegative with the Sabin–Feldman dye test, the modified agglutination test, and the Western blot. Results indicate that until examination of meat for T. gondii infection is implemented in slaughterhouses, all meat should be cooked according to industry guidelines before human consumption.

High prevalence and genotypes of Toxoplasma gondii isolated from goats, from a retail meat store, destined for human consumption in the USA

Little information is available concerning the presence of viable Toxoplasma gondii in tissues of goats worldwide. In the present study, hearts of 234 goats obtained from a local USA grocery store were examined for T. gondii infection. Blood clot or fluid removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Antibodies to T. gondii were found in 125 (53.4%) of 234 goats, with titers of 1:5 in 20, 1:10 in 44, 1:20 in 16, 1:40 in five, 1:160 in five, 1:320 in five, and 1:640 or higher in 30 goats. Hearts of 112 goats (46 goats <1:5, and 66 goats 1:10 or higher) were used for isolation of viable T. gondii by bioassays in mice. For bioassays, 50 g of the myocardium were digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. Toxoplasma gondii was isolated from 29 goats; from hearts of one of 46 with titers of <1:5, one of nine with titers of 1:10, one of three with titers of 1:40, and 26 of 40 with titers of 1:160 or higher. Two isolates were highly virulent to outbred Swiss Webster mice; all infected mice died of toxoplasmosis, irrespective of the dose. All T. gondii isolates were subsequently grown in cell cultures. Genotyping of the 29 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) from DNA obtained from cell culture grown tachyzoites revealed 12 genotypes. Nine isolates were clonal Type II lineage, four isolates had type II alleles at all loci except a type I allele at the Apico locus, and four isolates were clonal Type III. The remaining 12 strains were divided into nine atypical genotypes, including five new and four previously identified genotypes. DNA sequences of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) were generated for the five new genotypes. Comparing these sequences with previously published data revealed no unique sequences in these goat strains. Taken together, these results indicate high parasite prevalence and moderate genetic diversity of T. gondii in goats, which have important implications in public health. We believe this is the first genetic analysis of T. gondii isolates from goats in the USA.

Identification of a Sporozoite-Specific Antigen from Toxoplasma gondii

Abstract Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst–induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii–infected humans. Serum antibody to TgERP was detected in humans within 6–8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti–T. gondii IgM detected in sera, or <30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti–T. gondii IgM detected in sera, or >30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.

Unrecognized Ingestion of Toxoplasma gondii Oocysts Leads to Congenital Toxoplasmosis and Causes Epidemics in North America

BACKGROUND Congenital toxoplasmosis presents as severe, life-altering disease in North America. If mothers of infants with congenital toxoplasmosis could be identified by risks, it would provide strong support for educating pregnant women about risks, to eliminate this disease. Conversely, if not all risks are identifiable, undetectable risks are suggested. A new test detecting antibodies to sporozoites demonstrated that oocysts were the predominant source of Toxoplasma gondii infection in 4 North American epidemics and in mothers of children in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS). This novel test offered the opportunity to determine whether risk factors or demographic characteristics could identify mothers infected with oocysts. METHODS Acutely infected mothers and their congenitally infected infants were evaluated, including in-person interviews concerning risks and evaluation of perinatal maternal serum samples. RESULTS Fifty-nine (78%) of 76 mothers of congenitally infected infants in NCCCTS had primary infection with oocysts. Only 49% of these mothers identified significant risk factors for sporozoite acquisition. Socioeconomic status, hometown size, maternal clinical presentations, and ethnicity were not reliable predictors. CONCLUSIONS Undetected contamination of food and water by oocysts frequently causes human infections in North America. Risks are often unrecognized by those infected. Demographic characteristics did not identify oocyst infections. Thus, although education programs describing hygienic measures may be beneficial, they will not suffice to prevent the suffering and economic consequences associated with congenital toxoplasmosis. Only a vaccine or implementation of systematic serologic testing of pregnant women and newborns, followed by treatment, will prevent most congenital toxoplasmosis in North America.