Toxoplasmosis in a Hawaiian monk seal (Monachus schauinslandi)

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment with oocysts. T. gondii was identified in a Hawaiian monk seal (Monachus schauinslandi) that had visceral and cerebral lesions. Tachyzoites were found in the lymph nodes, spleen, diaphragm, heart, adrenal glands, and brain. A few tissue cysts were found in sections of the cerebrum. The diagnosis was confirmed serologically, by immunohistochemical staining with T. gondii-specific polyclonal rabbit serum, and by the detection of T. gondii DNA. The genotype was determined to be type III by restriction fragment length polymorphisms of the SAG2 gene. This is the first report of T. gondii infection in a Hawaiian monk seal.     

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.     

Meningoencephalitis associated with an unidentified apicomplexan protozoan in a Pacific harbor seal

A Pacific harbor seal (Phoca vitulina richardsii) was found on the central California coast with neurologic signs and labored breathing, which were unresponsive to treatment. Necropsy revealed a nonsuppurative necrotizing meningoencephalitis, a multilocular thymic cyst, and nonsuppurative cystitis and renal pyelitis. Microscopic examination revealed protozoans in the brain, thymic cyst, and bladder mucosa. Ultrastructurally, the protozoal tachyzoites were different from those of Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona; the rhoptries were small and had electron-dense contents, and the organism divided by endodyogeny. Specific antibodies were not detected in serum using agglutination (N. caninum, T. gondii) and immunoblot assays (S. neurona). Immunohistochemistry for these organisms was negative. Polymerase chain reaction on brain tissue using specific primers did not amplify T. gondii deoxyribonucleic acid. The meningoencephalitis in this seal thus appears to have been caused by a novel protozoan.     

Toxoplasma gondii antibodies in free-living African mammals.

Twelve species of free-living African mammals from Kenya, Tanzania, Uganda and Zambia were tested for antibodies to Toxoplasma gondii using the indirect hemagglutination test. Of 157 animals sampled, 20 (13%) were seropositive. T. gondii antibodies were detected in Burchell's zebra, (Equus burchelli), hippopotamus (Hippopotamus amphibius), African elephant (Loxodonta africana), defassa waterbuck (Kobus defassa), lion (Panthera leo), and rock hyrax (Procavia capensis), The highest titers were found in elephants, two having titers of 1:4096 and one of 1:8192. These results are discussed in relation to the maintenance of T. gondii among African wildlife.     

Seroprevalence of Toxoplasma gondii in Canadian pinnipeds

Sera (n = 328) collected from phocids (1995-97) from the east coast of Canada, including harp seals (Phoca groenlandica), hooded seals (Cystophora cristata), grey seals (Halichoerus grypus), and harbor seals (Phoca vitulina), were diluted 1:25, 1:50, and 1:500 and tested by a modified agglutination test for antibodies to Toxoplasma gondii. Titers equal to or greater than 1:25 were considered evidence of exposure. Grey seal (11/122, 9%), harbor seal (3/34, 9%), and hooded seal (1/60, 2%) had titers of 1:25 and 1:50. Harp seals (n = 112) were seronegative. Probable maternal antibody transfer was observed in one harbor and one grey seal pup at 10 and 14 day of age, respectively. Transmission of T gondii in the marine environment is not understood. The discovery of T. gondii in marine mammals might indicate natural infections unknown because of lack of study or might indicate recent contamination of the marine environment from the terrestrial environment by natural or anthropogenic activities.     

Toxoplasma gondii infections in red-tailed hawks inoculated orally with tissue cysts

The response to inoculation of Toxoplasma gondii tissue cysts was examined in 3 red-tailed hawks (Buteo jamaicensis). One hawk (hawk 1) was inoculated orally with 3.000 tissue cysts of the GT-1 isolate of T. gondii and 2 hawks (hawks 2 and 3) each were inoculated orally with 12,000 tissue cysts of a mixture of 8 isolates of T. gondii. None of the hawks developed clinical signs of toxoplasmosis. Serum antibodies were measured with the modified direct agglutination test using formalin-fixed tachyzoites. Hawk 1 had a titer of 1:40 prior to inoculation and did not have an increase in titer during the study. Hawks 2 and 3 had titers of 1:5 and 1:10, respectively, prior to inoculation, and both had increased titers (titers greater than or equal to 1:60) by 1 wk postinoculation and remained T. gondii antibody positive throughout the 10 wk of the study. Toxoplasma gondii was isolated from the heart and breast muscle of hawk 1. The biologic behavior of this T. gondii isolate was different from the 1 inoculated, and it probably represents a prior natural infection. Toxoplasma gondii was isolated from the brain, heart, breast muscle, and a mixture of gizzard and proventriculus from hawk 2 and from breast muscle of hawk 3. Toxoplasma gondii was not isolated from the eye, lung, liver, kidney, or spleen of any red-tailed hawk.     

Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Argentina

The prevalence of Toxoplasma gondii in free-ranging chickens can be considered a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 29 free-range chickens (Gallus domesticus) from Argentina was investigated. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 19 of 29 (65.5%) chickens. Hearts and brains of seropositive (MAT > or = 1:5) chickens were bioassayed in mice. Toxoplasma gondii was isolated from 9 of 19 seropositive chickens. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 1 was type I, 1 was type II, and 7 were type III. This is the first report of isolation of T. gondii from chickens from Argentina.     

Molecular and biologic characteristics of Toxoplasma gondii isolates from wildlife in the United States

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered more virulent in outbred mice and have been isolated predominantly from clinical cases of human toxoplasmosis, whereas types II and III isolates are considered less virulent for mice and are found in humans and food animals. Little is known of genotypes of T. gondii isolates from wild animals. In the present report, genotypes of isolates of T. gondii from wildlife in the United States are described. Sera from wildlife were tested for antibodies to T. gondii with the modified agglutination test, and tissues from animals with titers of 1:25 (seropositive) were bioassayed in mice. Toxoplasma gondii was isolated from the hearts of 21 of 34 seropositive white-tailed deer (Odocoileus virginianus) from Mississippi and from 7 of 29 raccoons (Procyon lotor); 5 of 6 bobcats (Lynx rufus); and the gray fox (Urocyon cinereoargenteus), red fox (Vulpes vulpes), and coyote (Canis latrans) from Georgia. Toxoplasma gondii was also isolated from 7 of 10 seropositive black bears (Ursus americanus) from Pennsylvania by bioassay in cats. All 3 genotypes of T. gondii based on the SAG2 locus were circulating among wildlife.     

Toxoplasma gondii Hsp70 as a danger signal in toxoplasma gondii-infected mice

Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-gamma knockout mice infected with T gondii. Tgondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.     

A survey of Neospora caninum and Toxoplasma gondii infection in urban rodents from Brazil

Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii , and a few findings indicated that they may be natural intermediate hosts for N. caninum . The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus , Rattus norvegicus , and Mus musculus captured in urban areas of S?o Paulo reveal a striking low frequency of occurrence of these infections.     

Isolation of Toxoplasma gondii from a naturally infected beef cow.

Toxoplasma gondii was isolated from the intestinal wall of an adult shorthorn cow. The cow had an antibody titer of 1:1,600 in the T. gondii agglutination test using formalin-fixed whole tachyzoites. The strain of the parasite designated CT-1 was lethal to mice. This is the first report of the recovery of viable T. gondii from a naturally infected cow in the United States.     

Attenuation of the virulent RH strain of Toxoplasma gondii by passages in mice immunized with Toxoplasma lysate antigens

The virulent RH strain of Toxoplasma gondii was attenuated after a few passages or just one long passage in mice immunized twice with a four-week interval between immunizations with an emulsion of Toxoplasma lysate antigens and complete Freund's adjuvant. Three avirulent strains, RH-cyst III, IV and VIII were established from the RH strain. The RH-cyst III strain was effective for vaccination against challenge with the original, virulent RH strain. The attenuation of T. gondii is an expression of the innate attributes of this parasite necessary to maintain its parasitic life cycle in nature.     

A heterophile antigen associated with experimental toxoplasmosis

The biological characteristics of a heterophile protein (HP) in peritoneal exudate from mice, hamsters, rats, and guinea pigs infected with Toxoplasma gondii were studied by immunofluorescence, immunoelectrophoresis and immunodiffusion techniques using specific antisera raised in rabbits. HP of mice had the highest antigenicity, HP of hamsters and rats had intermediate antigenicity and HP of guinea pigs had the lowest antigenicity. HP was found in normal peritoneal exudates from mice, hamsters and rats inoculated with paraffin oil instead of T. gondii and in normal guinea pig serum. HP was detected by the fluorescent antibody technique on the surface of T. gondii in peritoneal exudates of mice, but not on mouse peritoneal cells, and by the indirect fluorescent antibody technique on L cells infected with T. gondii and on free Toxoplasma derived from them, but not on uninfected L cells. T. gondii could make host cells produce HP to cover its surface for protection. The relation between HP from host cells and T. gondii is discussed.     

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.     

Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.     

Direct evidence for Toxoplasma gondii infection in a wild serow (Capricornis crispus) from mainland Japan

Toxoplasma gondii infection was studied in 41 Japanese serows ( Capricornis crispus ), a goat-antelope in mainland Japan. Blood and muscle specimens were collected from 41 subjects between 2006 and 2010. Presence of antibodies to T. gondii in the sera was examined by using the latex agglutination test (cutoff titer 1:32); 10 of 41 (24.4%) were seropositive. Toxoplasma gondii DNA was detected in muscle tissue of 1 seropositive serow using a semi-nested PCR assay for the B1 gene. A partial nucleotide sequence (220 bp) corresponding to the B1 gene of T. gondii was obtained by direct sequencing; the sequence was 99.1% identical to that of the RH strain. This study is the first report to show direct evidence for the T. gondii infection in Japanese serows.     

Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus)

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered virulent to outbred mice, whereas Type II and III isolates are not. In the present report, viable T. gondii was isolated for the first time from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), and black-winged lory (Eos cyanogenia). For the isolation of T. gondii, tissues were bioassayed in mice, and genotyping was based on the SAG2 locus. Toxoplasma gondii was isolated from 3 of 6 skunks, 1 of 4 Canada geese, and 2 of 2 feral cats (Felis catus) from Mississippi. All donor animals were asymptomatic. Viable T. gondii was also isolated from 5 of 5 lories that had died of acute toxoplasmosis in an aviary in South Carolina. Genotypes of T. gondii isolates were Type III (all skunks, lories, and the goose) and Type II (both cats). All 5 Type III isolates from birds and 2 of the 3 isolates from skunks were mouse virulent.     

Prevalence and isolation of Toxoplasma gondii from white-tailed deer in Alabama

Nineteen white-tailed deer (Odocoileus virginianus) from 5 counties in Alabama were examined for infection with Toxoplasma gondii. Twenty-gram samples of heart tissue were bioassayed in mice, serum was examined for T. gondii antibodies using the direct agglutination test, and sections of heart muscle were examined histologically for tissue cysts. Toxoplasma gondii was isolated from 4 of 19 (21%) white-tailed deer hearts. Antibody titers of greater than or equal to 1:50 were found in sera from 7 of 16 (44%) white-tailed deer. Histological examinations of tissue sections from white-tailed deer hearts were negative for T. gondii.     

Seroprevalence of Toxoplasma gondii in swine from slaughterhouses in Lima, Peru, and Georgia, U.S.A

Toxoplasma gondii is an important pathogen transmitted by food, with raw or undercooked meat as the main foodborne source of toxoplasmosis. In Peru, 2-4 million people have antibodies to T. gondii. It is believed that more than 60 million people in the United States are infected with T. gondii. In this study, the prevalence of T. gondii in pigs from Peru and the United States was determined by Western blot. The presence of IgG antibodies to T. gondii from serum samples was determined. Blood samples were collected from 137 pigs at a slaughterhouse in Lima, Peru, and 152 pigs at a slaughterhouse in Georgia. Of the serum samples collected from swine, 27.7% (n = 38) from Peru and 16.4% (n = 25) from the United States were positive for T. gondii. Swine represent a significant source of human infection with T. gondii in Peru and the United States.     

Endogenous polyamine levels in macrophages is sufficient to support growth of Toxoplasma gondii

Cytotoxic-activated macrophages control Toxoplasma gondii growth by producing nitric oxide (NO). However, the parasite can partially inhibit NO production. NO is generated from arginine within the polyamine biosynthetic pathway. Two enzymes of this pathway are ornithine, decarboxylase (ODC) and arginine decarboxylase (ADC). The aim of the present work was to investigate whether T. gondii is able to modulate polyamine metabolism in macrophages. Toxoplasma gondii infection did not affect basal ODC or ADC activity. However, lipopolysaccharide induced an increase in ODC activity. Polyamine-treated macrophages exhibited a T. gondii-infection index similar to controls but a higher adhesion index; the parasite did not grow in methyl-ornithine (ODC inhibitor)-treated macrophages. The parasites were able to take up putrescine with a Km of 0.92 microM, indicating the presence of a high-affinity putrescine-transporter system. Putrescine-treated T. gondii actively penetrated macrophages and Vero cells. However, NO production and lysosomal parasitophorous vacuole fusion were not inhibited. Considered together, these results demonstrate that T. gondii requires polyamines for multiplication. However, as opposed to Trypanosoma cruzi and because of a relatively high-affinity putrescine-transporter system in the parasite, constitutive macrophage levels of putrescine seem sufficient to support T. gondii survival and multiplication.