Toxoplasma gondii antibodies in naturally exposed wild coyotes, red foxes, and gray foxes and serologic diagnosis of Toxoplasmosis in red foxes fed T. gondii oocysts and tissue cysts

Antibodies to Toxoplasma gondii were determined in sera from 222 coyotes (Canis latrans), 283 red foxes (Vulpes vulpes), and 97 gray foxes (Urocyon cinereoargenteus) from Indiana, Kentucky, Michigan, and Ohio during 1990-1993. Sera were examined in 1:25, 1:100, and 1:500 dilutions by the modified direct agglutination test (MAT) with formalinized whole tachyzoites plus mercaptoethanol. Antibodies were found in 131 (59.0%) of 222 coyotes, 243 (85.9%) of 283 red foxes, and 73 (75.3%) of 97 gray foxes. Antibodies were also measured by different serologic tests in 4 littermate T. gondii-free red foxes fed T. gondii tissue cysts or oocysts; the fifth littermate fox was not fed T. gondii. Antibodies were measured in fox sera obtained 0, 14, and 36-55 days after infection with T. gondii. All 4 foxes fed T. gondii developed MAT and dye test antibody titers of 1:200 or more 14 days later. The latex agglutination test (LAT) and indirect hemagglutination test (IHAT) were less sensitive than MAT for the diagnosis of T. gondii infection in foxes. Antibodies were not detected by LAT (titer 1:64) in the 2 foxes fed tissue cysts nor by IHAT in 1 of the foxes fed tissue cysts. Toxoplasma gondii was isolated by bioassay in mice from tissues of all 4 foxes fed T. gondii. The control fox had no T. gondii antibodies detectable by any of the serologic tests.     

Tachyzoite-induced life cycle of Toxoplasma gondii in cats

The tachyzoite-induced cycle of Toxoplasma gondii was studied in 46 cats. Tachyzoites of the M-7741 or Me-49 strain of T. gondii were administered orally to cats by pouring into the mouth or by stomach tube, or by intraintestinal inoculation. Ten weaned cats that had been inoculated with tachyzoites directly in the intestine were killed 1, 3, 6, 9, 12, 15, 18, or 25 days later, and their tissues were studied histologically and bioassayed in mice. Toxoplasma gondii was demonstrable in the blood of 8 cats and in other tissues of all these 10. Four out of five 1- to 8-day-old cats fed tachyzoites by stomach tube became infected with T. gondii, and 1 became ill because of toxoplasmosis. All 19 weaned cats fed tachyzoites (poured into the mouth) became infected, and 6 died of acute toxoplasmosis 9-15 days after being fed T. gondii. Six out of 12 weaned cats fed tachyzoites by stomach tube became infected but were asymptomatic. Overall, 12 out of 26 cats observed for 19 days or more shed oocysts with a prepatent period (pp) of 19 days or more, with the sole exception of 1 cat that shed oocysts with a pp of 5 days. Enteroepithelial stages of T. gondii were not found in any cat before oocysts were shed. Cats shed up to 360 million oocysts in a day, and oocysts were shed for 4-6 days.     

Seroprevalence of Toxoplasma gondii in pregnant women and cats in Grenada, West Indies

Prevalence of antibodies against Toxoplasma gondii was studied in 534 pregnant women and 40 domestic cats in Grenada, West Indies. Antibodies (IgG) for T. gondii were sought in human sera by an enzyme-linked immunosorbent assay and in cat sera by using the modified agglutination test (MAT). Antibodies were found in 57 % of pregnant women. Seroprevalence increased with age; 51% of 15- to 19-yr-old women (100 total) had antibodies versus 60% of 20- to 24-yr-old women (127 total). Antibodies to T. gondii (MAT, 1:25 serum dilution) were found in 35% of cats; titers were 1:25 in 7 cats, 1:50 in 4 cats, and 1:500 in 3 cats. Epidemiological data suggested that the ingestion of food or water contaminated with oocysts was an important mode of transmission of T. gondii to women.     

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica)

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.     

Experimental Toxoplasma gondii infection in grey seals (Halichoerus grypus)

Laboratory-reared animals were used to assess the susceptibility of seals (Halichoerus grypus) to Toxoplasma gondii infection. Four seals were each orally inoculated with 100 or 10,000 oocysts of T. gondii (VEG strain), and another 4 seals served as negative controls. Occasionally, mild behavioral changes were observed in all inoculated seals but not in control animals. A modified agglutination test revealed the presence of antibodies to T. gondii in sera collected from inoculated seals and mice inoculated as controls. No evidence of the parasite was found on an extensive histological examination of seal tissues, and immunohistochemical staining of tissue sections from inoculated seals revealed a single tissue cyst in only 1 seal. Control mice inoculated with 10 oocysts from the same inoculum given to seals became serologically and histologically positive for T. gondii. Cats that were fed brain or muscle tissue collected from inoculated seals passed T. gondii oocysts in feces. This study demonstrates that T. gondii oocysts can establish viable infection in seals and supports the hypothesis that toxoplasmosis in marine mammals can be acquired from oocysts in surface water runoff and sewer discharge.     

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal

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 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.     

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.     

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from free-range chickens from Guatemala

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 Guatemala was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 37 (74%) chickens with titers of 1:5 (11), 1:10 (7), 1:20 (11), 1:40 (1), 1:80 (1), 1:160 (3), 1:1,280 (2), and 1:2,560 (1). Hearts, pectoral muscles, and brains of 19 chickens with MAT titers of 1:20 or more were bioassayed individually in mice. Tissues from the remaining 31 chickens with titers of 1:10 or lower were pooled and fed to 4 T. gondii-free cats (13 chickens with titers of less than 1:5 to 1 cat, 11 chickens with titers of 1:5 to 2 cats, and 7 chickens with titers of 1:10 to 1 cat). Feces of cats were examined for oocysts; they did not shed oocysts. Toxoplasma gondii was isolated from 8 chickens with MAT titers of 1:20 or more (from 1 of 11 chickens with a titer of 1:20 and all 7 chickens with a titer of 1:80 or more) from the heart, brain, and pectoral muscle (3); heart and pectoral muscle (1); and heart alone (4). Genotyping of these 8 isolates with the SAG2 locus indicated that 5 were type III and 3 were type 1. This is the first report of isolation of T. gondii from chickens from Guatemala.     

Characterization of Toxoplasma gondii isolates in free-range chickens from Argentina

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 61 free-range chickens (Gallus domesticus) from provinces of Santiago del Estero and Entre Rios, Argentina was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and were found in 25 chickens; titers were 1:5 in 6 chickens, 1:10 in 1 chicken, 1:20 in 2 chickens, 1:40 in 1 chicken, 1:80 in 2 chickens, 1:60 in 4 chickens, 1:120 in 2 chickens, 1:640 in 3 chickens, and 1: 1,280 or higher in 4 chickens. Hearts, pectoral muscles, and brains of 22 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 39 chickens with titers of 1:5 or less were pooled and fed to 3 T. gondii-free cats. Feces of cats were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 17 of 22 chickens with MAT titers of 1:10 or higher. Genotyping of these 17 isolates using polymorphisms at the SAG2 locus indicated that 4 were Type I, 3 were Type II, and 10 were Type III. Toxoplasma gondii isolates (2 Type I and I Type III) from 3 chickens were virulent for mice and 1 Type I was not mouse virulent. Prevalence of T. gondii antibodies in chickens varied among regions, being 3 times greater in the humid Pampeana region (61.2%) than in the semiarid plain of Santiago del Estero (20%).     

Prevalence of Toxoplasma gondii in chickens from an area in southern Brazil highly endemic to humans

The prevalence of Toxoplasma gondii in free-range chickens from Campos dos Goytacazes, Rio de Janeiro State, Brazil, was examined to evaluate environmental contamination by oocysts. Antibodies against T. gondii were assayed by the modified agglutination test (MAT) in sera of chickens. Antibodies against the parasite were found in 129 of 198 chickens with MAT titers > or = 1:25. Brains and hearts of 86 of the 198 chickens were bioassayed in mice for the presence of T. gondii. Viable parasites were isolated from 61 (70.9%) of the 86 chickens. Importantly, viable T. gondii were recovered even from seronegative chickens (MAT titer < or = 1:10). The distribution of parasite-positive chickens by MAT titer was 4 of 17 (titer < or = 1:10), 3 of 4 (titer of 1:20), 2 of 6 (titer of 1:40), and 52 of 59 (titer > or = 1:80). Thus, the high recovery rate of T. gondii observed in mice is indicative of high levels of environmental contamination of free-range chickens by T. gondii oocysts in this area that is endemic to humans.     

Isolation and molecular characterization of Toxoplasma gondii from chickens from Sri Lanka

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 100 free-range chickens (Gallus domesticus) from Sri Lanka was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 39 chickens with titers of 1:5 in 8, 1:10 in 8, 1:20 in 4, 1:40 in 5, 1:80 in 5, 1:160 in 5, 1:320 in 2, 1:640 or more in 2. Hearts and brains of 36 chickens with MAT titers of 1:5 or more were bioassayed in mice. Tissues of 3 chickens with doubtful titers of 1:5 were pooled and fed to a cat; the cat shed T. gondii oocysts in its feces. Tissues from 61 chickens with titers of less than 1:5 were pooled and fed to 2 T. gondii-free cats; the cats did not shed oocysts. Toxoplasma gondii was isolated from 11 of 36 seropositive chickens by bioassay in mice. All 12 T. gondii isolates were avirulent for mice. Genotyping of 12 isolates using the SAG2 locus indicated that 6 were type III, and 6 were type II. This is the first report of genetic characterization of T. gondii from any host in Sri Lanka.     

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.     

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.     

Toxoplasma gondii infections in chickens from Venezuela: isolation, tissue distribution, and molecular characterization

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 46 free-range chickens (Gallus domesticus) from Venezuela was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 16 (32%) chickens with titers of 1:5 in 1, 1:10 in 2, 1:40 in 2, 1:80 in 2, 1:160 in 2, 1:320 in 3, 1: 640 in 2, and 1:1,280 or higher in 2. Hearts, pectoral muscles, and brains of 13 chickens with MAT titers of 1:40 or more were bioassayed individually in mice. Tissues of each of 3 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice. Tissues from the remaining 30 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts; it did not shed oocysts. Toxoplasma gondii was isolated from 12 of 13 chickens with MAT titers of 1:40 or more. Toxoplasma gondii was isolated from pooled tissues of 1 of 2 chickens with titers of 1:10. Eight of these 13 isolates were virulent for mice. Genotyping of 13 of these isolates using the SAG2 locus indicated that 10 were type III, and 3 were type II. Phenotypically and genetically these isolates were different from T. gondii isolates from North America and Brazil. This is the first report of isolation of T. gondii from chickens from Venezuela.     

Seroprevalence of Toxoplasma gondii in harbor seals (Phoca vitulina) in southern Puget Sound, Washington.

As part of the Puget Sound Ambient Monitoring Program of the Washington Department of Fish and Wildlife, serum samples from 380 harbor seals (Phoca vitulina) were tested for antibodies to Toxoplasma gondii in the modified agglutination test (MAT) incorporating formalin-fixed tachyzoites and mercaptoethanol. Antibodies to T. gondii were found in 29 of 380 (7.6%) seals with titers of 1:25 in 13, 1:50 in 14, and > or = 1:500 in 2 seals. Results indicate natural exposure of these wild marine mammals to T. gondii oocysts.     

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.     

Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in the South American opossum (Didelphis marsupialis) from the city of São Paulo, Brazil

Antibodies to Neospora caninum and Toxoplasma gondii were assayed in sera of 396 opossums (Didelphis marsupialis) from the city of S?o Paulo, Brazil. Antibodies to N. caninum were assayed using the indirect immunofluorescent antibody test (IFAT). Antibodies (IFAT, approximately 1:25) to N. caninum were found in 84 opossums (D. marsupialis) in titers of 1:25 in 46, 1:50 in 20, 1:100 in 17, and 1:400 in 1. Antibodies to T. gondii were assayed with the modified agglutination test (MAT) and the IFAT. Antibodies to T. gondii (MAT, approximately 1:25) were found in 82 (20.4%) of the 396 opossums, in titers of 1:25 in 24, 1:50 in 26, 1:100 in 18, 1:200 in 13, and 1:800 in 1. The IFAT antibodies to T. gondii were found in 148 of 396 opossums, in titers of 1:16 in 41, 1:32 in 23, 1:64 in 13, 1:128 in 6, 1:256 in 20, 1:512 in 17, 1:1,024 in 10, 1:2,048 in 10, 1:4,096 in 7, and 1:8,192 in 1. This is the first report of N. caninum and T. gondii infections in D. marsupialis.     

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.     

Experimental toxoplasmosis in budgerigars (Melopsittacus undulatus)

The susceptibility of budgerigars (Melopsittacus undulatus) to graded doses of Toxoplasma gondii oocysts was studied. Sixteen budgerigars were divided into 4 groups (A-D) of 4 each. Birds in groups A-C were fed 100,000, 1,000, or 100 infective oocysts of the VEG strain of T. gondii, respectively. Budgerigars in group D were not fed oocysts and served as controls. All 4 birds in group A died (or were killed) because of acute severe enteritis 5 or 6 days after feeding oocysts (DAFO). Three of the 4 birds in group B were killed (or died) because of toxoplasmosis 9 or 14 DAFO. One budgerigar in group C and the 4 budgerigars in group D remained healthy and were killed 35 or 39 DAFO. Toxoplasma gondii was demonstrated in tissues of all budgerigars fed oocysts. The control budgerigars remained clinically normal and showed no evidence of T. gondii exposure. These results indicate that, compared to other passerines, budgerigars are relatively resistant to clinical toxoplasmosis.     

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.