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A female adult barred owl (Strix varia) had been hurt by a car. Its general status declined gradually within 2 wk with anorexia and inactivity. Necropsy examination revealed marked multifocal pale areas in liver, emaciation, and mild airsacculitis and pericarditis. Histopathologic examination revealed severe acute multifocal hepatic necrosis with numerous protozoal tachyzoites within necrotic foci and in the cytoplasm of hepatocytes and macrophages. These tachyzoites stained with an indirect immunohistochemistry method for Toxoplasma gondii antigens. This is the first reported case of hepatitis resulting from toxoplasmosis in a raptor.

To evaluate the efficacy of fetal serology in the diagnosis of bovine neosporosis abortion, sera from 48 fetuses with immunohistochemically confirmed neosporosis and 42 fetuses without demonstrable Neospora caninum were examined in the indirect fluorescent antibody test (IFAT). Fetal sera were diluted 2-fold starting at a 1:25 dilution. Antibodies to N. caninum were detected in 31 of 48 (65%) fetuses with confirmed neosporosis; the IFAT antibody titers were 1:25 (5 fetuses), 1:50 (17 fetuses), 1:200 (6 fetuses), andor = 1:800 (3 fetuses). Neospora caninum antibodies were found in 3 of 42 fetuses without demonstrable protozoa; in all 3 cases, a high titer was found suggesting undiagnosed congenital neosporosis. A recombinant antigen enzyme-linked immunosorbent assay was not useful for the detection of fetal antibodies to N. caninum.

Sarcocystis buffalonis n. sp. is proposed for a species forming thick-walled, macroscopic sarcocysts in skeletal muscles and the esophagus of the water buffalo (Bubalus bubalis). Sarcocysts of S. buffalonis were found in 68 (10.5%) of 647 buffalo carcasses examined grossly at slaughter in Ho Chi Minh City in southern Vietnam. Sarcocystis buffalonis sarcocysts were 1-8 mm long and 0.1-0.5 mm wide. The cyst wall was 3-7.7 microns thick and had palisadelike villar protrusions that were constricted at the base, expanded laterally in the mid-region, and tapered distally. The villar protrusions contained microfilaments and electron-dense granules. Sarcocysts of Sarcocystis fusiformis, the other well-known macroscopic species occurring in water buffalo, were also found in 60 of the 68 animals infected with S. buffalonis. Sarcocysts of S. fusiformis were thin walled and had characteristic cauliflowerlike villar protrusions. Two of 7 cats fed isolated S. buffalonis sarcocysts were found to have 12 x 8 microns sporocysts in their intestine or feces 10 days after inoculation.

Toxoplasma gondii oocysts are highly infective to intermediate hosts including humans, pigs, and mice, but are considered less infective for cats, the definitive host. To determine infectivity of T. gondii oocysts for cats, 20 2- to 3-mo-old T. gondii-free cats in groups of 4 were fed graded doses of oocysts estimated to have 1, 10, 100, 1,000, or 10,000 mouse infective oocysts of the VEG strain of T. gondii. Feces of cats were examined for at least 35 days after feeding oocysts. All cats were killed, necropsied, their sera were tested for T. gondii antibodies, and tissues were bioassayed in mice. Three of the 4 cats fed 10,000 oocysts, 3 of the 4 cats fed 1,000 oocysts, and 2 of the 4 cats each fed 100 oocysts shed 7.3-162 million T. gondii oocysts in their feces, with a prepatent period of 18-44 days. Based on bioassay and antibody production, all 4 cats fed 10,000 oocysts, 3 of 4 cats fed 1,000 oocysts, 2 of 4 cats fed 100 oocysts, and 0 of 8 cats fed 1 or 10 oocysts acquired T. gondii infection. Antibodies to T. gondii were detected by the modified agglutination test in all 9 bioassay-proven T. gondii-infected cats and in none of the 11 cats without demonstrable T. gondii. In a series of other experiments, the age of the cat at the time of oocyst feeding and the administration of corticosteroids were found to have no influence on the prepatent periods after ingestion of oocysts. A review of published and unpublished data indicated that the minimum prepatent period to shedding of oocysts after the ingestion of oocysts by cats is 18 days.

To follow antibody responses measured by various serologic tests in pigs orally inoculated with low (or = 10 oocysts) numbers of Toxoplasma gondii oocysts.24, 2- to 3-month-old pigs.Pigs (n = 42) were inoculated orally with 10 (14 pigs) or 1 (28 pigs) infective oocysts, and 6 pigs served as uninoculated controls. Blood (serum) samples were obtained at 1- to 3-week intervals until euthanasia. At necropsy, the brain, heart, and tongue of pigs were bioassayed in mice and cats for isolation of T gondii. Modified agglutination test (MAT), using whole, fixed tachyzoites and mercaptoethanol; latex agglutination test (LAT); indirect hemagglutination test (IHAT); Sabin-Feldman dye test (DT); and ELISA were used to evaluate serologic responses to T gondii.T gondii was isolated from tissues of 13 of 14 pigs each fed 10 oocysts, 17 of 28 pigs each fed 1 oocyst, and 0 of 6 control pigs. 29 of 30 T gondii-infected pigs developed antibodies when measured by MAT, DT, and ELISA; the 1 seronegative-infected pig had been fed 10 oocysts and was euthanatized 69 days after inoculation. LAT detected antibodies in 26 of 30 T gondii-infected pigs. IHAT detected antibodies in 11 T gondii-infected pigs.MAT, DT, and ELISA were more sensitive serologic assays than LAT and IHAT for detecting antibodies induced by low numbers of T gondii in pigs.

Rats are considered to be 1 of the most resistant hosts for Toxoplasma gondii infection, but relative infectivity of T. gondii for rats is not known. Therefore, infectivity and pathogenicity of oocysts of the VEG strain of T. gondii were studied in Sprague Dawley weaned rats (approximately 130 g). Groups of 5 rats were each inoculated orally with 1 to 1 million infective oocysts. Three of the 5 rats fed 1 million oocysts died of acute toxoplasmosis between 6 and 9 days after ingesting oocysts; all other rats survived. Tissue cysts were found in brains of all rats fedor = 10 oocysts and in 3 of 6 rats fed 1 oocyst. The average number of tissue cysts in brains of rats was 300, 180, 528, 600, 396, 1,200, and 2,650 in rats fed 1, 10, 100, 1,000, 10,000, 100,000 or 1 million oocysts, respectively. Microscopic lesions were seen in brains of all T. gondii-infected rats and the frequency of lesions was usually proportional to the dose. Antibodies (or = 1:512) to T. gondii were detected in sera of all infected rats 29 days after ingestion of oocysts by the modified agglutination test, the commercially available latex agglutination test, and the indirect hemagglutination test.

Toxoplasma gondii oocysts are highly resistant to environmental influences. To study the effect of alpha-irradiation on the viability of T. gondii oocysts, 1 million sporulated oocysts of the VEG strain were irradiated to absorbed doses of 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, or 0.50 kGy at 5 C with a 137Cs gamma-irradiation source. Treated oocysts were bioassayed for viable T. gondii in mice. Viable T. gondii was not found in brains of mice 2 mo after oral inoculation with oocysts irradiated at levelsor = 0.25 kGy. Therefore, alpha-irradiation is an effective means of killing T. gondii oocysts. Mice inoculated orally with oocysts irradiated at 0.20 and 0.40 kGy were partially protected when challenged orally with lethal doses of nonirradiated oocysts.

To evaluate antigen recognition patterns of serum IgM, IgG, and IgA from queens and their kittens as a method of diagnosing neonatal toxoplasmosis.5 pregnant queens were inoculated orally with Toxoplasma gondii tissue cysts during gestation (18 to 44 days). On various days after parturition (0 to 97), serum was obtained from queens and kittens (n = 19).Tissues from most kittens were assessed for T gondii infection by bioassay in mice. Serum samples were evaluated by IgM, IgG, and IgA western blot immunoassays for the presence of T gondii antibodies. Antigens recognized by kitten serum samples, but not by the corresponding queen serum sample, were considered to indicate neonatal infection with T gondii.Using the results of western blot immunoassay, 8 of 19 kittens (age, 2 to 97 days) were determined to be infected with T gondii. Western blot immunoassay results correlated well with bioassay results, identifying 7 of 8 bioassay-positive kittens. Western blot immuno-assay additionally identified 1 kitten as infected, but tissues from the kitten had not been bioassayed. In each of the 5 kittens that developed clinical signs of toxoplasmosis, the diagnosis of neonatal toxoplasmosis was supported by results of the western blot immunoassays.Comparison of queen and kitten T gondii antigen recognition patterns of IgM, IgG, and IgA can be used for antemortem diagnosis of neonatal toxoplasmosis.

Antibodies to Neospora caninum were detected in 5 (10%) of 52 coyotes from Texas. Antibodies to Toxoplasma gondii were detected in 32 (62%) of 52 samples from these same coyotes. Four (80%) of the 5 coyotes that were seropositive for N. caninum also had antibodies to T. gondii. Nineteen (37%) of the coyotes did not have antibodies to either parasite. Three coyote pups were inoculated with the brains from mice infected with 3 strains of N. caninum originally isolated from dogs. None of the pups developed neosporosis or excreted N. caninum oocysts in their feces. The pups developed anti-N. caninum antibody titers ofor = 1:800 but did not develop antibodies to T. gondii. Results of this study indicate that antibodies to T. gondii are more common than antibodies to N. caninum in coyotes. Additionally, young coyotes appear to be resistant to experimental N. caninum infection.

To define the infectiousness of the VEG strain of Toxoplasma gondii, 42 pigs were fed doses estimated at 10, 1, or1 mouse infective oocysts. They were killed 38-99 days after inoculation and 50 g of tissues from their tongue, heart, and brain were individually homogenized in acidic pepsin solution and bioassayed in mice. Pools of brain, heart, tongue, and skeletal muscle (total 500 g) were bioassayed in cats. Toxoplasma gondii was isolated by bioassays in mice and in cats from 13 of 14 pigs fed 10 oocysts, 13 of 14 pigs fed 1 oocyst, and 4 of 14 pigs fed "less than" 1 oocyst, indicating high infectivity of VEG strain of T. gondii to pigs. All infected pigs developed modified agglutination test antibodies (1:50). Control pigs (n = 6) remained seronegative (1:20) and T. gondii was not isolated from their tissues. Toxoplasma gondii was isolated from tongues of 27 (93%), brains of 21 (72%), and hearts of 13 (45%) of 29 experimentally infected pigs by bioassay in mice. The number of T. gondii-positive mice after inoculation of tongue, brain, and heart from infected pigs was 240 (80%), 84 (28%), and 36 (12%) of 300 mice inoculated with each organ, respectively. Thus, the VEG strain of T. gondii was localized more often and in higher numbers in the tongue than in the brain and the heart of pigs. The apparent muscle localization after infection with the low dose of the VEG strain of T. gondii agrees with other studies in livestock that suggest T. gondii is more neurotropic in mice than in livestock.

An unidentified Toxoplasma gondii-like disseminated infection was found in tissues of a 2-day-old hartebeest. Protozoal tachyzoites were about 3.5 x 1.7 Microns, had electron-dense rhoptries, and divided by endodyogeny. The organism did not stain with polyclonol antibodies to T. gondii, Neospora caninum, or Sarcocystis cruzi.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.

Neospora caninum tissue cysts were found in sections of the brain from a full-term stillborn deer of Eld (Cervus eldi siamensis) from a zoo in France. There was N. caninum-associated nonsuppurative encephalitis and the diagnosis was confirmed in immunohistochemical staining with antibodies specific to N. caninum.

A Sarcocystis neurona-like organism was associated with granulomatous encephalitis in an ataxic male juvenile striped skunk (Mephitis mephitis) from Cape Cod, Massachusetts. Various stages of schizonts and merozoites of S. neurona were seen within some of the granulomata.

Sera from 1,367 white-tailed deer (Odocoileus virginianus) from 4 geographic regions in Minnesota collected during 4 hunting seasons (1990-1993) were tested for antibodies to Toxoplasma gondii using the modified direct agglutination test incorporating mercaptoethanol. Sera from 30% of the deer had antibody titersor = 25; 8.6% were positive at a titer of 25, 11% at a titer of 50, and 10% at a titeror = 500. There was a significant increase in seropositivity with age (P0.0001). Adult deer were twice as likely to be positive as yearlings; yearlings were 2.5 times as likely to be positive as fawns. There was no difference in prevalence by sex when adjusted for age (P = 0.316), nor was there age-sex interaction. Only males showed a slight increase in titer with age (P = 0.049). There were no significant differences in prevalence among the regions of northeast pine/aspen forest, southwest tall-grass prairie, southeast mixed-hardwood forest, and aspen/oak suburban park land. There were no statistically significant differences by year of collection. The prevalence of T. gondii antibodies in white-tailed deer remains high and deer hunters and consumers should ensure that venison is well-cooked or frozen prior to consumption.

An adult porcupine (Coendou mexicanus) from the National Zoo, San Jose, Costa Rica, died because of toxoplasmosis involving the heart, lungs, liver, and kidneys. Toxoplasma gondii was found in lesions and the diagnosis was confirmed by immunohistochemical staining with T. gondii-specific polyclonal rabbit antibodies. This is a new host record for T. gondii.

Neospora canium tachyzoites and tissue cysts were found in tissues of a goat fetus aborted after 3.5 months of gestation. The fetus had hydrocephalus and a hypoplastic cerebellum. The predominant lesion in the fetus was severe encephalitis associated with numerous N canium tissue cysts. Parasites in fetal tissues reacted positively with N caninum antibodies in immunohistochemical tests. The doe was clinically normal and had a 1:800 antibody titer to N caninum as determined by use of an indirect fluorescent antibody test 9 months after abortion. Five of 77 other does from this herd also had indirect fluorescent antbody titers to N caninum that wereor = 1:100.

As part of a long-term study on immunity to oocyst shedding, 12 4-6-mo-old cats were inoculated orally with tissue cysts of the ME-49 strain (6 cats) or the TS-2 strain (6 cats) of Toxoplasma gondii. Two cats fed the ME-49 strain died or were killed because of acute toxoplasmosis 12 and 13 days after inoculation (DAI), respectively. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. One cat died following rechallenge infection due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Blood samples were obtained weekly or monthly and sera were analyzed for antibodies to T. gondii using the modified agglutination test (MAT), the Sabin-Feldman dye test (DT), and the enzyme-linked immunosorbent assay (ELISA) for IgM (IgM-ELISA) or IgG (IgG-ELISA). The MAT was performed using both formalin-fixed (FF) and acetone-fixed (AF) tachyzoites. The MAT (FF) was the most sensitive test; cats seroconverted within 14 DAI and high titers (10,000) persisted6 yr, although cats had no clinical signs. The MAT titers using the AF detected recent exposure and titers declined sharply after 2 mo postinoculation. DT and ELISA titers were lower and developed slower than MAT titers. Fluctuations in antibody titers were limited to 8-fold during the 6-yr observation period. Anamnestic serum antibody responses were seen in 2 cats after the final challenge, but not after first challenge.

Sarcocystis neurona is an apicomplexan that causes equine protozoal myeloencephalitis (EPM) in North and South America. Horses appear to be an aberrant host, because the merozoites continually divide in the central nervous system, without encysting. The natural host species has not previously been identified. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was compared to those of Sarcocystis muris, Sarcocystis cruzi, Toxoplasma gondii, and Cryptosporidium parvum to identify a unique region suitable for a species-specific amplification primer. The S. neurona SSURNA primer was used in a polymerase chain reaction (PCR) assay for the purpose of identifying this organism in feces and intestinal digest of wildlife specimens. Sporocysts were isolated from 4 raccoons (Procyon lotor), 2 opossums (Didelphis virginiana), 7 skunks (Mephitis mephitis), 6 cats (Felis catus), 1 hawk (Accipiter sp.), and 1 coyote (Canis latrans). The S. neurona SSURNA PCR assay and a control PCR assay using protist-specific primers were applied to all sporocyst DNA samples. All sporocyst DNA samples tested positive on the control assay. The SSURNA PCR assay yielded a 484-bp product only when applied to opossum samples. The SSURNA gene of both opossum sporocyst samples was sequenced to determine its relationship to the S. neurona SSURNA gene. The sequence had 99.89% similarity with S. neurona. This suggests that opossums are the definitive host of S. neurona.

Immunity to Toxoplasma gondii, as measured by oocyst shedding, was studied in cats. In 3 trials, 12 3-mo-old T. gondii-free cats were fed tissue cysts of the ME-49 strain of T. gondii. All cats shed T. gondii oocysts for approximately 1 wk starting 3-5 days after ingesting tissue cysts. One cat became ill because of toxoplasmic pneumonia and was killed 17 days after inoculation. The remaining cats remained clinically normal. Approximately 3 mo after primary infection, these 11 cats (immune) and 11 age-matched or littermate uninfected cats (nonimmune) were challenged orally with tissue cysts of the ME-49 strain. In trials 1 and 3, 1 immune and 1 nonimmune cat were killed at 36 hr, 60 hr, 5 days, and 12 days after challenge and the development of T. gondii in intestines was studied histologically; in trial 2, cats were killed at 36 hr, 60 hr, and 5 days only. None of the "immune" cats shed oocysts after challenge. Asexual T. gondii types were found at 36 and 60 hr and at 5 days, indicating partial development of T. gondii in the intestine of immune cats. There were no significant differences in lymphocyte CD4+/CD8+ from spleen, popliteal, and mesenteric lymph nodes of immune cats compared to nonimmune cats.

Two epidemiologic studies of risk factors for transmission of Toxoplasma gondii to swine were conducted for farms in Illinois. The first study was a cross-sectional survey of swine farms from the state of Illinois pseudorabies testing program, in which farm owners or managers were interviewed by telephone regarding presence of risk factors for transmission of T. gondii on the farm. There were 123 farms surveyed that provided blood samples for at least 30 sows. The mean sow seroprevalence was 19.5% (median = 10.0%). Multiple regression analysis of the association of sow seroprevalence with outdoor housing of sows, cat access to sow areas, number of sows, open feed storage and water delivery, delayed removal of carcasses, and presence of rodents on the farm indicated that higher sow seroprevalence was associated with cat access to sows (P = 0.009) and fewer sows in the herd (P = 0.05). The second study was a field investigation of 47 swine farms (37 from the cross-sectional study). Data collection included obtaining blood samples from swine, cats, and rodents, and fecal samples from cats, heart and brain tissue from rodents, and feed, water, and soil samples for T. gondii examination. The risk of T. gondii transmission from cats and rodents to sows and finishing pigs was evaluated, taking into account housing conditions and herd size. Multiple regression analysis indicated that T. gondii seroprevalence in finishing pigs increased with more seropositive juvenile cats on the farm (P0.0001) and higher seroprevalence in house mice (P = 0.0023).(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental oral inoculations to evaluate potential definitive hosts of Neospora caninum were conducted by feeding infected rodent tissues to 9 carnivorous birds of 4 species. Birds included 2 red-tailed hawks (Buteo jamaicensis), 2 turkey vultures (Cathartes aura), 2 barn owls (Tyto alba), and 3 American crows (Corvus brachyrhynchus). The rodents (mice or rats) had been inoculated with 100,000 culture-derived tachyzoites of N. caninum 1-6 mo before feeding to the birds. Fecal samples were collected from each bird daily for 1 mo after feeding rodents and examined for oocysts by fecal flotation. In addition, processed aliquots from all avian fecal samples were fed to BALB/c mice. Five weeks after feeding, mice were bled and sera were tested for antibodies against N. caninum. One to two months later, mice were killed and brain tissue was examined microscopically for protozoal cysts. While occasional oocysts were found in avian fecal samples, these were likely not N. caninum because they were not infective to BALB/c mice. It was concluded that the bird species tested are not likely to be definitive hosts of N. caninum.

Herein we report a murine model to examine transplacental transmission, transmammary transmission, or both, of Neospora caninum. Prevalence of transplacental transmission in outbred Swiss-Webster mouse pups was 85%, with 11 of 13 litters containing at least 1 transplacentally infected pup. Sixty-two percent of litters born to experimentally infected dams contained 85% or more transplacentally infected pups. Numbers of pups congenitally infected per litter was higher if dams were inoculated during the first half of pregnancy. Transplacental transmission decreased to 25% when a singly infected dam delivered a second litter. Transmammary transmission was observed in 1 of 51 pups suckling dams experimentally infected 5, 10, or 15 days postparturition. No pups were infected when cross-fostered onto chronically infected dams.

Field studies were conducted on 47 swine farms in Illinois during 1992 and 1993 to identify sources and reservoirs of Toxoplasma gondii infection. Blood samples were obtained from swine and from trapped wildlife. Serum antibodies to T. gondii were determined using the modified agglutination test, incorporating mercaptoethanol. Antibodies to T. gondii (titeror = 25) were found in 97 of 4,252 (2.3%) finishing pigs, 395 of 2,617 (15.1%) sows, 267 of 391 (68.3%) cats, 126 of 188 (67.0%) raccoons, 7 of 18 (38.9%) skunks, 29 of 128 opossums (22.7%), 6 of 95 (6.3%) rats, 3 of 61 (4.9%) white-footed mice (Peromyscus sp.), and 26 of 1,243 (2.1%) house mice (Mus musculus). Brains and hearts of rodents trapped on the farm were bioassayed in mice for the presence of T. gondii. Toxoplasma gondii was recovered from tissues of 7 of 1,502 (0.5%) house mice, 2 of 67 (3.0%) white-footed mice, and 1 of 107 (0.9%) rats. Feces of 274 cats trapped on the farm and samples of feed, water, and soil were bioassayed in mice for the presence of T. gondii oocysts. Toxoplasma gondii was isolated from 2 of 491 (0.4%) feed samples, 1 of 79 (1.3%) soil samples, and 5 of 274 (1.8%) samples of cat feces. All mammalian species examined were reservoirs of T. gondii infection. All farms had evidence of T. gondii infection either by detection of antibodies in swine or other mammalian species, or by detection of oocysts, or by recovery from rodents by bioassay. The possibility of transmission of T. gondii to swine via consumption of rodents, feed, and soil was confirmed.

Neospora caninum-induced abortion is a major production problem in the dairy cattle industry in the United States and worldwide. Abortions attributable to naturally acquired N caninum infection also have been observed in pygmy goats. We studied experimentally induced infections with N caninum in pregnant pygmy does to determine whether abortions attributable to N caninum infection would occur after inoculation. Seven pregnant pygmy does (1 control doe and 6 inoculated with N caninum) were studied. The control doe remained clinically normal throughout the study and delivered 2 healthy kids. Abortion, fetal death, and stillbirths were observed in some pregnant does inoculated with N caninum. Two pregnant pygmy does inoculated with N caninum early in gestation (day 51) had fetuses that died and were aborted, or died and were reabsorbed. Neospora caninum tachyzoites and lesions were observed in the brain, spinal cord, and heart of aborted fetuses; parasites also were isolated from the placenta. Four additional pregnant pygmy does (2 inoculated at mid-gestation [day 85], and 2 at late gestation [day 127]) did not abort after inoculation. However, 1 doe inoculated during mid-gestation delivered a stillborn fetus that had died about 1 week prior to parturition. This kid was congenitally infected with N caninum. Neospora caninum was isolated from the placentas of all inoculated does examined. Neonatal neosporosis was not observed in live-born kids, nor were stages of N caninum isolated from any live-born kid. Does did not undergo abortion or have congenitally infected kids when they were rebred and evaluated for neosporosis.

The sensitivity and specificity of various serologic tests for antibodies to Toxoplasma gondii were compared in 1,000 naturally exposed sows, using isolation of viable T gondii as the definitive test. Serum samples obtained from heart blood of 1,000 sows from Iowa were examined for T gondii antibodies by use of the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and ELISA. Toxoplasma gondii was isolated from 170 hearts of 1,000 sows by bioassays in mice and cats. The percentage of samples diagnosed as positive for each of the serologic tests was: MAT = 22.2% (titeror = 1:20), IHAT = 6.4% (titeror = 1:64), LAT = 10.4% (titeror = 1:64), and ELISA = 24.1% (OD0.360). The sensitivity and specificity of these tests were calculated respectively to be: 82.9 and 90.29% for MAT, 29.4 and 98.3% for IHAT, 45.9 and 96.9% for LAT, and 72.9 and 85.9% for ELISA. The dye test was run at 1:20 dilution on only 893 sera because of bacterial contamination and presence of anticomplement substances. Dye test antibodies were found in 17.8% of the sera, and sensitivity and specificity were 54.4 and 90.8%, respectively. Thus, the MAT had the highest sensitivity among all serologic tests used.

The nuclear small subunit ribosomal RNA (nss-rRNA) gene sequence of Neospora spp. isolated from cattle was analyzed and compared to the sequences from several closely related cyst-forming coccidial parasites. Double-stranded DNA sequencing of 5 bovine Neospora spp. isolates (BPA1-4), 2 Neospora caninum isolates (NC-1 and NC-3), and 3 Toxoplasma gondii isolates (RH, GT-1, CT-1) were performed and compared to each other, as well as to other sequences available in GenBank for the NC-1 isolate, Sarcocystis muris, and Cryptosporidium parvum. There were no nucleotide differences detected between the Neospora spp. isolates from cattle and dogs. Four nucleotide differences were consistently detected when sequences of Neospora spp. isolates were compared to those of the T. gondii isolates. These results indicate that Neospora spp. and T. gondii are closely related, but distinct, species.

Sixteen pregnant queens were inoculated orally with tissue cysts of Toxoplasma gondii, and fetal membranes and offspring were examined for T gondii infection by bioassay in mice. Queens appeared clinically normal, although all shed T gondii oocysts. Toxoplasma gondii was isolated from tissues of 7 of 33 fetuses or kittens from 5 litters (at 13, 23, 26, 27, and 29 postinoculation days) from 8 queens euthanatized between 10 and 31 postinoculation days. Infection with T gondii was found in kittens from all 8 litters from the 8 queens that were allowed to undergo parturition and nurse their kittens. A total of 43 kittens were born to these 8 queens. Toxoplasma gondii was isolated from tissues of 26 of 40 kittens bioassayed; in 3 kittens, tissues were not available for bioassay. Toxoplasmosis was severe in full-term kittens born to 5 queens; all 25 kittens from these litters died or were ill by 24 days of age. Anorexia, lethargy, hypothermia, and sudden death were the most common manifestations. Cytologic examination of peritoneal fluid aspirate samples and determination of hepatic-associated enzyme concentrations in affected kittens, as well as measurement of anti-T gondii antibodies in serum of kittens and queens, were helpful in the diagnosis of neonatal toxoplasmosis. Transplacental transfer of anti-T gondii antibodies was not observed in cats. Toxoplasma gondii oocysts were found in fecal samples of 3 kittens from different litters at 16, 24, and 63 days of age.

Cats that have shed Toxoplasma gondii oocysts are considered to be immune to reshedding of oocysts. To investigate if this immunity persists in cats for 6 yr, 12 4-6-mo-old cats without T. gondii antibodies were inoculated orally with tissue cysts of the ME-49 strain (6 cats) and the TS-2 strain (6 cats) of T. gondii. All of them shedor = 20 million oocysts between 4 and 13 days after feeding tissue cysts. Two cats became ill between 11 and 13 days after primary infection; 1 died on the 13th day, and the other had to be killed on the 11th day because of generalized acute toxoplasmosis. Toxoplasma gondii oocysts were not found on the hair of 10 cats examined 7 days after cats had shed millions of oocysts. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. None of the challenged cats shed oocysts. One cat died due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Four of these 9 cats re-shed T. gondii oocysts; 3 of them had been challenged also at 39 days after primary infection. Two control cats housed together with chronically infected cats for 6 yr remained seronegative for T. gondii; both of these shed oocysts after challenge with the P89 strain.

A serologic survey that tested for antibodies to Toxoplasma gondii was conducted, using the modified direct agglutination test, on 6,965 serum samples collected from swine in 179 herds in Illinois in 1992. In breeding swine, results for 1,057 of 5,080 (20.8%) sera tested were positive. In growing/finishing swine, results for 59 of 1,885 (3.1%) sera tested were positive, which was substantially lower than the seroprevalence rate estimated in a serosurvey of pigs from abattoirs in Illinois in 1983 and 1984. Data in the survey reported here were summarized for herds having at least 28 samples/herd. Among all herds, the median, mean, and maximum seroprevalence rates were 6.7, 16.1, and 96.8%, respectively, for breeding swine in 172 herds, and 0.0, 2.8, and 20.0%, respectively, for growing/finishing pigs in 44 herds. Among the 172 herds with breeding swine, 61 (35.5%) had no seropositive pigs. Among the 44 herds with growing/finishing swine, 28 (63.6%) had no seropositive pigs. A logistic regression model was used to estimate that the cumulative risk of T gondii infection for swine in herds containing seropositive pigs was 9.0% by 6 months of age for a herd that had the median seroprevalence rate. In contrast, for pigs in herds in the upper quartile of seroprevalence rates, risk of infection by 6 months of age was estimated to be greater than 20%. Analysis of these data would suggest that overall prevalence of T gondii infection in pigs from Illinois is low; nevertheless, there is a small proportion of farms for which the rate of T gondii infection in swine is moderately high.

Neospora caninum was identified in a lung aspirate specimen from an adult dog with severe pneumonia. Neosporosis was tentatively diagnosed by cytologic examination of a Wright-Giemsa-stained smear of the aspirate specimen, using light microscopy. The infection was confirmed by immunohistochemical examination of a section of lung tissue obtained at necropsy. Neosporosis is usually a fatal ascending neurologic disease of dogs less than 1 year of age. Neospora caninum infections are uncommon in adult dogs and do not have a consistent clinical course. The disease in the dog of this report was unique because the dog had clinical respiratory tract disease and because preliminary diagnosis was made by cytologic examination.

Neospora caninum is a protozoan parasite that causes severe disease in transplacentally infected dogs and abortions in domestic ruminants. Rodent models of neosporosis rely on treatment of hosts with methylprednisolone acetate (MPA) to enhance infections. The present study reports the development of an inbred BALB/c mouse model that results in central nervous system neosporosis in the absence of MPA treatment. Seven of 12 BALB/c mice died 26-70 days after subcutaneous (s.c.) inoculation with tachyzoites of the NC-1 strain of N. caninum, and none of 12 BALB/c mice died after s.c. inoculation with tachyzoites of the NC-3 strain. None of 8 HSD:ICR mice (4 mice, NC-1 strain; 4 mice, NC-3 strain) developed clinical neosporosis or died after s.c. inoculation with tachyzoites. Control BALB/c (2) and HSD:ICR (2) mice s.c. inoculated with Hanks' balanced salt solution did not develop clinical signs of disease. Some mice in all N. caninum-inoculated groups had brain lesions, but significantly (P0.05) more BALB/c mice inoculated with the NC-1 strain had brain lesions.

Serum samples from 593 white-tailed deer in Pennsylvania that were killed by hunters in 1991 were examined for Toxoplasma gondii antibodies, by use of the modified agglutination test. Sixty percent (357/593) of the deer had T gondii antibodies; 10% had titer of 25, 23% had titer of 50, and 27% had titeror = 500. Sex-specific differences in prevalence were not detected.

Serum samples from 545 woodchucks Marmota monax from 22 counties in Pennsylvania were examined for antibodies to Toxoplasma gondii by the modified direct agglutination test. Fifty-one woodchucks (9.4%) had antibodies to T. gondii, with 10% at dilutions of 1:25, 2% at dilutions of 1:50, and 4% at dilutions of 1:500. This is the first report of T. gondii antibodies in woodchucks.

Hearts of 1,000 pigs killed at an abattoir in Iowa were bioassayed for the prevalence of tissue cysts of Toxoplasma gondii. One hundred grams of cardiac muscle from each pig was homogenized, digested in pepsin solution, and bioassayed in 10 mice. Five hundred grams of heart tissue from each of a subset of 183 pigs was also bioassayed in cats. Serum collected from the heart from each pig was assayed for anti-T. gondii antibodies in the modified agglutination test using formalin-fixed whole tachyzoites. Anti-T. gondii antibodies were found in 22.2% of pigs. Viable T. gondii was isolated from a total of 170 pigs; from 50 hearts by bioassay in mice, from 58 hearts by bioassay in both mice and cats, and from 62 pigs by bioassay in cats only. The success of isolation in cats (65.6%) was approximately twice that in mice (31.7%). Percentage of isolations of T. gondii with respect to reciprocal antibody titers (in parentheses) in pigs was: 3.7% (20), 37.1% (20), 38.1% (40), 60% (80), 75% (200), 77% (400), 83% (800), and 75.8% (or = 2,000 to 16,000).

During the hunting season of 1993, hearts from 28 black bears (Ursus americanus) from Pennsylvania were examined for Toxoplasma gondii infection. Serum samples from heart blood were examined for T. gondii antibodies by the use of the modified agglutination test (MAT), the Sabin-Feldman dye test (DT), the latex agglutination test (LAT), and the indirect hemagglutination test (IHA). One-hundred grams of myocardium from each bear were bioassayed in mice. Approximately 500 g of myocardium from 11 bears were fed to 11 cats, and feces from those cats were examined for T. gondii oocysts. Toxoplasma gondii MAT antibodies (or = 1:40) were found in 22 bears in titers of 1:40 (4 bears), 1:80 (3 bears), 1:160 (7 bears), 1:320 (4 bears), andor = 1:2,000 (4 bears). Antibodies to T. gondii by DT (or = 1:10), LAT (or = 1:32), and IHA (or = 1:64) tests were found in 21, 9, and 6 bears, respectively. All 6 bears that did not have T. gondii antibodies in MAT were also negative in DT, IHA, and LAT. Viable T. gondii was isolated from 3 bears by bioassays in mice and from an additional 7 bears by bioassays in cats. All 10 bears that had viable T. gondii had MAT and DT antibodies but only 6 and 5 of them had antibodies by LAT and IHA, respectively.

Sarcocystis neurona is a coccidial parasite that causes a neurologic disease of horses in North and South America. The natural host species are not known and classification is based on ultrastructural analysis. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was amplified using polymerase chain reaction techniques and sequenced by Sanger sequencing reactions. The sequence was compared with partial sequences of S. muris, S. gigantea, S. tenella, S. cruzi, S. arieticanis, S. capracanis, Toxoplasma gondii, Eimeria tenella, and Cryptosporidium parvum. Alignments of available sites for all 10 species and alignments of the entire SSURNA sequence of S. neurona, S. muris, S. cruzi, T. gondii, and C. parvum were performed. Alignments were analyzed using maximum parsimony and maximum likelihood methods to determine relative phylogeny of these organisms. These analyses confirmed placement of S. neurona in the genus Sarcocystis and suggested a close relationship to S. muris, S. gigantea, and T. gondii. Molecular phylogeny suggests that Sarcocystis spp., which utilize the dog (Canis familiaris) as the definitive host, evolved from a common ancestor, whereas those species (including T. gondii) that utilize the cat (Felis domesticus) as the definitive host evolved from another common ancestor. This suggests a possible definitive host for S. neurona.

A murine monoclonal antibody (MAB) 6G7 generated against tachyzoites of Neospora caninum recognized 8 major and several minor antigens, as observed by western blot analysis. Relative rate of migration of the 8 major antigens ranged from 31 to 97.4 kd. In addition, MAB 6G7 recognized a Toxoplasma gondii tachyzoite antigen with a relative rate of migration of 107 kd. Immunogold labeling of N caninum tachyzoites grown in human foreskin fibroblast cells indicated that MAB 6G7 binds to micronemes, dense granules, basal portions of rhoptries, and intravacuolar tubules within the parasitophorous vacuole. Monoclonal antibody 6G7 also bound to micronemes and basal portions of rhoptries within tachyzoites of T gondii. Monoclonal antibody 6G7 did not significantly inhibit development of tachyzoites in vitro.

To study the effect of gamma-irradiation on the viability of Toxoplasma gondii tissue cysts, brains of mice inoculated with 95 newly isolated strains of T. gondii from pigs and 10 other laboratory isolates were pooled, flattened, packed in vacuum, and irradiated to absorbed doses of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.9 kGy at 5 C (+/- 0.5 C) with a 137Cs gamma-radiation source. Treated samples were bioassayed for viable T. gondii in mice, cats, or both. Tissue cysts of all strains were rendered nonviable at 0.4 kGy. To study the effect of temperature on inactivation by irradiation, tissue cysts were irradiated at -4, 0, 4, 8, 12, and 16 C (+/- 0.5 C) at 0.25 kGy. Temperature during irradiation had no marked effect on the viability of tissue cysts.

Neospora caninum causes serious disease in dogs, and it, or a similar parasite, is a major cause of abortion in cattle. Little is known about the susceptibility of this protozoan to antimicrobial agents. We studied several antimicrobial agents to determine which classes might have activity against this parasite. We also determined whether activity of such agents was coccidiocidal or coccidiostatic. A 2-day of treatment, monoclonal antibody-based enzyme immunoassay and a 5-day of treatment, cell culture flask (CCF), lesion-based assay were developed to examine the ability of test agents to inhibit tachyzoite multiplication. Seven sulfonamides were examined, with the following activities observed: sulfathiazoleor = sulfamethoxazolesulfadiazinesulfaquinoxalineor = sulfamethazinesulfadimethoxinesulfamerazine. Dapsone, a sulfone, had little activity. Six dihydrofolate reductase/thymidylate synthase inhibitors were examined, with the following activities observed: piritreximpyrimethamineormetoprimtrimethoprim = diaveridinemethotrexate. Six ionophorous antibiotics were examined; lasalocid, maduramicin, monensin, narasin, and salinomycin had equivalent activities, but alborixin was toxic for host cells at the lowest concentration examined. Three macrolide antibiotics--azithromycin, clarithromycin, and erythromycin--were examined and had equivalent activities. Two tetracycline antibiotics, doxycycline and minocycline, were examined and had equivalent activities. Three lincosamide antibiotics were examined, with the following activities observed: clindamycin hydrochlorideclindamycin phosphatelincomycin hydrochloride. Pentamidine and 6 of its analogs were examined, and only hexamidine and 1,4-Di[4-(2-imidazolinyl)-2-methoxy-phenoxy]butane had activity. Eight miscellaneous antiprotozoal agents were examined for activity. Amprolium, metronidazole, paromomycin, and roxarsone had little activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The safety of vaccination and persistence and distribution of Toxoplasma gondii stages within tissues following vaccination were examined in 3-day-old nursing pigs vaccinated with living tachyzoites by intravenous and subcutaneous routes of either the TS-4 mutant strain or its parent RH strain of T. gondii. The efficacy of vaccination of nursing pigs with the TS-4 mutant was also examined in pigs challenged orally with oocysts following vaccination. Pigs were vaccinated with 3 x 10(5) living tachyzoites when 3 days old and boosted with 3 x 10(5) living tachyzoites when 17 days old. Group 1 had 2 pigs vaccinated intravenously (i.v.) with Hanks' balanced salt solution (HBSS) and served as a vaccination control. Group 2 and 5 pigs vaccinated i.v. with tachyzoites of the TS-4 mutant; 3 pigs were used to examine the safety, persistence, and distribution of the TS-4 mutant and 2 were used for oocyst challenge. Group 3 had 5 pigs vaccinated i.v. with tachyzoites of the RH strain and all were used to examine the safety, persistence, and distribution of the RH strain within their tissues. Group 4 had 3 pigs vaccinated subcutaneously (s.c.) with tachyzoites of the TS-4 mutant; 1 was used to determine the persistence and distribution of the TS-4 mutant within its tissues and the other 2 pigs were used for GT-1 oocyst challenge studies. Group 5 had 3 pigs vaccinated s.c. with tachyzoites of the RH strain and all were used to examine the safety, persistence, and distribution of the RH strain within their tissues. None of the control pigs or pigs vaccinated with the TS-4 mutant developed clinical signs of disease or died prior to oocyst challenge. The TS-4 mutant was not reisolated from the tissues of vaccinated pigs nor were microscopic lesions present in the tissues of pigs that had been killed and examined at necropsy. Severe disease with clinical signs consisting of dyspnea, inactivity, diarrhea, and ocular lesions was observed in the group 3 pigs vaccinated i.v. with the RH strain. One pig died 7 days after initial vaccination. Microscopic lesions were observed in numerous tissues of all group 3 pigs. Swelling, erythema, and ulcers were observed at the site of inoculation in the group 5 pigs that were vaccinated s.c. with the RH strain. Minimal to no microscopic lesions were observed in these group 5 pigs. The RH strain was reisolated from pigs in both groups vaccinated with this strain.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatozoon canis-like schizonts were found in the hearts of 2 lionesses (Panthera leo) from Kenya. The parasite, initially described as a new genus and new species Microbesnoitia leoni Bwangamoi, 1989, is determined to be a junior synonym of Hapatozoon canis (James, 1905) Wenyon, 1926.

Fourteen 2-3-wk-old turkeys were inoculated orally with 10(5) or 10(4) infective oocysts of the ME 49 strain of Toxoplasma gondii. Of the 8 turkeys given 10(5) oocysts in experiment 1, 3 died or were killed 12 or 14 days after inoculation (DAI) because of respiratory distress associated with a concomitant Aspergillus-like fungus infection. The remaining 5 turkeys remained normal and were killed 62 DAI. Toxoplasma gondii was isolated in mice from the heart of all 5, from the breast muscles of 2, leg muscles of 3, and from the brains and livers of none of the turkeys. All 6 turkeys fed 10(4) oocysts in experiment 2 remained clinically normal until necropsy on 41 DAI; T. gondii was isolated from pooled tissues from each turkey. All 14 turkeys developed high antibody titers to T. gondii in the modified agglutination test (MAT) using formalinized tachyzoites. The enzyme-linked immunosorbent assay (ELISA) was as sensitive as MAT for detecting T. gondii antibodies in turkey sera. The latex agglutination and indirect hemagglutination tests were less sensitive than the MAT and ELISA. No dye-test-measurable antibodies were found in sera of any turkey.

Toxoplasmosis was diagnosed histologically in 9 kittens and 1 queen from 5 liters. In litter 1, four 3-month-old Siamese kittens and the queen were affected. The queen died of generalized toxoplasmosis, and her kittens died or were euthanatized 20, 22, or 28 days later. In litter 2, two of 3 Abyssinian 4- and 4.5-month-old kittens died of toxoplasmosis. In litter 3, an Abyssinian, delivered by cesarean section, became ill 17 days after delivery, and died 2 days later because of toxoplasmic hepatitis and pneumonia. In litter 4, three kittens, approximately 1 month old, were shedding Toxoplasma gondii-like oocysts, and the organism was identified histologically in tissues of 1 of them. In litter 5, one 3-week-old kitten out of 4 became ill. Toxoplasma gondii-like oocysts were found in feces and T gondii organisms were found in histologic sections of tissues.

Tissue sections from 119 cats that died or were euthanatized (1952-1990) because of toxoplasmosis-like illness were reexamined for Toxoplasma gondii by direct microscopy and immunohistochemical staining with anti-T gondii serum. Clinical and pathologic data from 100 of these cats with histologically verified toxoplasmosis were then analyzed. Of these 100 cats, 36 were considered to have generalized toxoplasmosis, 26 predominantly pulmonary lesions, 16 abdominal, 2 hepatic, 1 pancreatic, 1 cardiac, 2 cutaneous, 7 neurologic, and 9 had neonatal toxoplasmosis. In 14 cats, concurrent microbial infections or other maladies were seen. Cats were 2 weeks to 16 years old (median, 2 years; mean, 4 years). Sixty-five cats were males and 34 were females; sex was not recorded for 1 cat. Of 67 cats that had rectal temperatures recorded, 49 (73%) had fever (40.0 to 41.7 C). Dyspnea, polypnea, and signs of abdominal discomfort were frequently observed. Toxoplasmosis had been confirmed antemortem in 8 cats; 4 had a serum antibody titer to T gondii ofor = 1:1,024; and T gondii had been found in cytologic evaluation of tracheal aspirates from 2 cats and pleural fluid from 1 cat, as well as in a biopsy specimen of a mesenteric lymph node from another. Of the 15 cats with T gondii serum-antibody titers determined by the Sabin-Feldman dye test, 6 had no antibody detected in 1:4 dilution of their serum. Indirect fluorescent antibody titers were found in 10 of 10 cats' sera tested. Forty-one eyes from 27 of the cats were examined microscopically. Twenty-two of the 27 cats (81.5%) had evidence of intraocular inflammation in one or both eyes. Multifocal iridocyclochoroiditis was the most common lesion and was seen in 18 (81.8%) of the cats with ophthalmitis. The ciliary body was the most often severely affected portion of the uvea. Of the 22 cats with ocular toxoplasmosis, T gondii was found in eyes of 10. Toxoplasma gondii was found in the retina of 5 cats, the choroid of 2, the optic nerve of 1, the iris of 3, and the ciliary body of 4. Toxoplasma gondii was identified in 80% of 55 brains, 70.0% of 90 livers, 76.7% of 86 lungs, 64.4% of 45 pancreata, 62.7% of 59 hearts, 45.8% of 72 spleens, 41.5% of 65 intestines, 17.7% of 61 kidneys, and 60.0% of 30 adrenal glands.

Parasite-specific antibody responses were detected using an indirect fluorescent antibody (IFA) test in cattle that were naturally or experimentally infected with Neospora parasites. The test was developed using Neospora tachyzoites isolated from an aborted bovine fetus and grown in bovine cell cultures (isolate BPA1). In all cases, infections were confirmed by the identification of Neospora tachyzoites and/or bradyzoite cysts in fetal or calf tissues using an immunoperoxidase test procedure. Fifty-five naturally infected cows that aborted Neospora-infected fetuses had titers of 320-5,120 at the time of abortion. The titer of 6 cows that were serologically monitored over a prolonged period decreased to 160–640 within 150 days after they aborted infected fetuses. Two of the cows showed an increase in their Neospora titers during their subsequent pregnancy, and they gave birth to congenitally infected calves that had precolostral titers of 10,240-20,480. Postcolostral titers of these calves and of 4 other calves with congenital Neospora infections were all 25,120, whereas calves with no detectable parasites had titers ≤ 160. Two pregnant heifers that were experimentally infected with the BPA1 isolate at approximately 120 days gestation seroconverted to Neospora antigens within 9 days and developed peak titers of 5,120 and 20,480 within 32 days of infection. The fetus taken by caesarian section 32 days postinfection from 1 heifer and the full-term calf born to the other had Neospora titers of 640 and 10,240, respectively. Nine cows that aborted uninfected fetuses and 61 adult cattle maintained under pasture or feedlot conditions, where risk of exposure to Neospora was considered to be low, had titers ≤ 320. Some of the feedlot cattle tested had serologic reactivity that was restricted to antigens at the apical end of both Neospora and Toxoplasma gondii tachyzoites. This type of reactivity, which may result from serologic cross-reactivity between conserved apical complex antigens of closely related sporozoan parasites, differed from the whole parasite fluorescence that was observed with sera from Neospora-infected animals. The significance of these results and the potential application of the IFA test for the diagnosis of Neospora infections in cattle are discussed.

Ultrastructure of the interactions of host cell mitochondria with developing Toxoplasma gondii tissue cysts was examined in cultured cells, using transmission electron microscopy of infected cells and rhodamine 123 (a mitochondria-specific vital fluorescent dye) staining of isolated tissue cysts. Structurally mature T gondii tissue cysts were observed as early as 2 days after inoculation of cultured cells. During development of T gondii, host cell mitochondria were observed surrounding the parasitophorous vacuole membrane. Mitochondria became flat and elongated in the vicinity of the parasitophorous vacuole membrane. These mitochondria were also closely associated with T gondii tissue cysts. Incubation of tissue cysts from cultured cells and tissue cysts from mouse brains with rhodamine 123 revealed fluorescence of the tissue cyst wall in living specimens. Incubation of tissue cysts with 10 microM rotenone caused diminished fluorescence of the tissue cyst walls, and 100 microM rotenone caused complete inhibition. Mouse RBC, and tissue cysts fixed in 100% methanol did not fluoresce after exposure to rhodamine. Tissue cysts in 9 isolates of T gondii from mouse brains were examined, using rhodamine 123, and the tissue cysts walls of all isolates fluoresced, indicating no isolate effects. Our results indicate that host cell mitochondria may be closely associated with the tissue cysts of T gondii in cell cultures and in mice.

We determined the extent of the serologic cross-reactivity in the indirect fluorescent antibody (IFA) test for Babesia gibsoni, and the optimal cut-off titer for seropositivity in the test. The lowest titer to B gibsoni detected in a dog with naturally acquired clinical babesiosis was 1,280, and 7 of 12 dogs had titer between 10,240 and 20,480. Two experimentally infected normosplenic dogs developed high titer (40,960 to 81,920) to B gibsoni, and the same sera reacted in IFA tests for B canis (titeror = 640), Toxoplasma gondii (titeror = 2,560), and Neospora caninum (titeror = 10,240). Dogs that were experimentally infected with B canis and T gondii had titeror = 160 to B gibsoni. Dogs that were experimentally infected with N caninum had titer (80 to 10,240) to N caninum, but failed to have serologic reactivity to B gibsoni. Serologic titer of healthy dogs from Australia, a country where B gibsoni is not known to exist, wasor = 160 to B gibsoni. On the basis of these findings, a cut-off titer of 320 was considered to be appropriate for serodiagnosis of B gibsoni in dogs with clinical signs of babesiosis. A more conservative titer of 1,280 was established as the cut-off titer for seroepidemiologic studies based on relative costs and benefits of false-positive results and failure to isolate B gibsoni parasites after splenectomy and immunosuppression from a clinically normal dog with B gibsoni titer of 640.(ABSTRACT TRUNCATED AT 250 WORDS)