O.C.H. Kwok

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.

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and Sarcocystis canis-like infections in marine mammals

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and S. canis are related protozoans that can cause mortality in many species of domestic and wild animals. Recently, T. gondii and S. neurona were recognized to cause encephalitis in marine mammals. As yet, there is no report of natural exposure of N. caninum in marine mammals. In the present study, antibodies to T. gondii and N. caninum were assayed in sera of several species of marine mammals. For T. gondii, sera were diluted 1:25, 1:50, and 1:500 and assayed in the T. gondii modified agglutination test (MAT). Antibodies (MAT > or =1:25) to T. gondii were found in 89 of 115 (77%) dead, and 18 of 30 (60%) apparently healthy sea otters (Enhydra lutris), 51 of 311 (16%) Pacific harbor seals (Phoca vitulina), 19 of 45 (42%) sea lions (Eumetopias jubatus) [corrected] 5 of 32 (16%) ringed seals (Phoca hispida), 4 of 8 (50%) bearded seals (Erignathus barbatus), 1 of 9 (11.1%) spotted seals (Phoca largha), 138 of 141 (98%) Atlantic bottlenose dolphins (Tursiops truncatus), and 3 of 53 (6%) walruses (Odobenus rosmarus). For N. caninum, sera were diluted 1:40, 1:80, 1:160, and 1:320 and examined with the Neospora agglutination test (NAT) using mouse-derived tachyzoites. NAT antibodies were found in 3 of 53 (6%) walruses, 28 of 145 (19%) sea otters, 11 of 311 (3.5%) harbor seals, 1 of 27 (3.7%) sea lions, 4 of 32 (12.5%) ringed seals, 1 of 8 (12.5%) bearded seals, and 43 of 47 (91%) bottlenose dolphins. To our knowledge, this is the first report of N. caninum antibodies in any marine mammal, and the first report of T. gondii antibodies in walruses and in ringed, bearded, spotted, and ribbon seals. Current information on T. gondii-like and Sarcocystis-like infections in marine mammals is reviewed. New cases of clinical S. canis and T. gondii infections are also reported in sea lions, and T. gondii infection in an Antillean manatee (Trichechus manatus manatus).

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.

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, Paraná 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-induced murine toxoplasmosis: life cycle, pathogenicity, and stage conversion in mice fed Toxoplasma gondii oocysts.

The development of sporozoites to tachyzoites and bradyzoites was studied in mice after feeding 1-7.5 x 10(7) Toxoplasma gondii oocysts. Within 2 hr after inoculation (HAI), sporozoites had excysted and penetrated the small intestinal epithelium. At 2 HAI, most sporozoites were in surface epithelial cells and in the lamina propria of the ileum, and by 8 HAI, T. gondii was also seen in mesenteric lymph nodes. At 12 HAI, sporozoites had divided into 2 tachyzoites in the lamina propria of the small intestine. By 48 HAI, there was a profuse growth of tachyzoites in the intestine and mesenteric lymph nodes of mice fed 7.5 x 10(7) oocysts. Parasites had disseminated via the blood and lymph to other organs by 4 days after inoculation (DAI). Toxoplasma gondii was first isolated from peripheral blood at 4 HAI. Tissue cysts were visible histologically in the brain at 8 DAI. By using immunohistochemical staining with anti-bradyzoite-specific (BAG-5 antigen) serum, BAG-5-positive organisms were first seen at 5 DAI in the intestine and at 8 DAI in the brain. Using the bioassay in cats, bradyzoites were first detected in mouse tissues between 6 and 7 DAI, and they were found in intestines before they were found in the brain. Cats fed murine tissues containing bradyzoites shed oocysts in their feces with a short (< 10 days) prepatent period, whereas cats fed tissues containing tachyzoites did not shed oocysts within 3 wk. Using a pepsin-digestion procedure and mouse bioassay, bradyzoites were first detected in brain tissue at 7 DAI and in many organs of mice at 51 and 151 DAI. Individual bradyzoites, small and large tissue cysts, and tachyzoites were seen in the brains of mice at 87 and 236 DAI.

COMPLETION OF THE LIFE CYCLE OF SARCOCYSTIS NEURONA

Sarcocystis neurona is the most important cause of a neurologic disease in horses, equine protozoal myeloencephalitis (EPM). The complete life cycle of S. neurona, including the description of sarcocysts and intermediate hosts, has not been completed until now. Opossums (Didelphis spp.) are definitive hosts, and horses and other mammals are aberrant hosts. In the present study, laboratory-raised domestic cats (Felis domesticus) were fed sporocysts from the intestine of a naturally infected opossum (Didelphis virginiana). Microscopic sarcocysts, with a maximum size of 700 × 50 µm, developed in the muscles of the cats. The DNA of bradyzoites released from sarcocysts was confirmed as S. neurona. Laboratory-raised opossums (D. virginiana) fed cat muscles containing the sarcocysts shed sporocysts in their feces. The sporocysts were ∼10–12 × 6.5–8.0 µm in size. Gamma interferon knockout mice fed sporocysts from experimentally infected opossums developed clinical sarcocystosis, and S. neurona was identified in their tissues using S. neurona-specific polyclonal rabbit serum. Two seronegative ponies fed sporocysts from an experimentally-infected opossum developed S. neurona-specific antibodies within 14 days.

Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.

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.