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Coccidiosis is a common disease of livestock, including water buffaloes. Reports discussing the prevalence and identity of coccidian parasites in water buffaloes from Egypt are few, and nothing is known concerning the most pathogenic species

Coccidiosis is a common disease of camels, and camels are important for the economy of Asia and the Arabian Peninsula. Little is known regarding the prevalence of coccidian parasites in camels in Egypt. Fecal samples collected from the rectums of 200 camels at the Cairo slaughterhouse were processed using the sucrose flotation technique.

Toxoplasmosis in wild turkeys (

The coccidian parasite

Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-γ gene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk ( Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 post-inoculation (PI). The KO mice developed neurological signs and were necropsied 38-54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (1 μm thick) and smooth. Ultrastructural details of sarcocysts are described.

The life cycle of Sarcocystis species is heteroxenous (2-host), with carnivores being the definitive host and herbivores serving as intermediate hosts in predator-prey relationships. Raptors (eagles, hawks, falcons, and owls) are apex predators and are not consumed routinely by other carnivores, making the occurrence of sarcocysts in their muscles unusual. Recent reports of sarcocysts in eagles and owls with Sarcocystis encephalitis suggests that this condition may be becoming more frequent, and Sarcocystis falcatula has been implicated as the agent of encephalitis in golden ( Aquila chrysaetos) and bald eagles ( Haliaeetus leucocephalus) as well as great horned owls ( Bubo virginianus). The present study was done to determine the prevalence of sarcocysts of Sarcocystis species in the muscles of raptors from the southeastern United States. Pectoral and heart muscle from 204 raptor patients from the Carolina Raptor Center, Huntersville, North Carolina were tested for the presence of Sarcocystis species using histology. Only a few sarcocysts were seen in sections of pectoral muscle from 39 of 204 raptors (19.1%) and heart muscle from 9 that also had sarcocysts in their pectoral muscle. Two structural types of sarcocysts, thin-walled (1 μm; 62%) or thick-walled (2 μm, 38%), were seen. Statistical analysis of raptor age and gender was done by Fisher's exact test on samples from raptors with 20 or more samples per group. The prevalence of sarcocysts by age (2 yr or more) was significant for red-shouldered hawks ( Buteo lineatus) ( P = 0.022) and Cooper's hawks ( Accipiter cooperii) ( P = 0.028). Sarcocyst prevalence in male raptors from these groups evaluated statistically were always less than in females. Prevalence in female red-tailed hawks ( Buteo jamaicensis) (42.1%) was significantly greater than in males (6.7%) using Fisher's exact test ( P = 0.047). Examination of case histories from the 39 sarcocyst-positive raptors did not reveal an association with sarcocysts in raptor pectoral or heart muscle and in a diagnosis of encephalitis. Additional studies are needed to determine the epidemiology and relationships of Sarcocystis spp. that use raptors as intermediate hosts and the importance of Sarcocystis spp. in the overall wellbeing of raptors in their natural environments.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generation of schizonts have been reported in the small intestine of dogs before oocysts are excreted 9-11 days post inoculation (PI). Here, we re-evaluated asexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria throughout the small intestine. Merozoites of different sizes were present, often in the same vacuole. They were arranged singly, in pairs, and many within a single parasitophorous vacuole. The maximum number of nuclei within developing merozoites in a group was 8, but it could not be discerned if they were individual nuclei or parts of merozoites. Findings of abundant asexual stages 1 day after dogs had started excreting oocysts indicated continued asexual multiplication beyond the prepatent period. The stages found resemble the 3 generations reported previously. The mode of division of the asexual generations remains unclear. The results of the present study indicate that there are many generations that are difficult to determine because of the multiplication of merozoites in the original host cell without leaving it to enter new host cells. From the literature, it is evident that cat and dog coccidia ( Cystoisospora spp.) divide by more than 1 type of division, including endodyogeny. In the past, the schizont/meront groups containing more than 1 generation have been called "cysts." However, cyst is not an accurate term because it is best used for an orally infective stage of coccidia; monozoic tissue cysts of C. canis can occur in paratenic hosts in extraintestinal organs. We recommend the term "types" as originally proposed for intestinal stages of Toxoplasma gondii and used for the original description of the life cycle of C. suis of swine when describing endogenous stages of the Sarcocystidae. Ultrastructural studies are needed to determine the precise form of multiplication of canine Cystoisospora species.

Toxoplasma gondii is a significant worldwide parasitic protozoan. In the present study, prevalence of antibodies of T. gondii was examined from 29 free-ranging black bears (Ursus americanus) from south-central Florida where the host species was listed as state threatened during this project. Overall T. gondii prevalence was found to be 44.8%, specifically 46.2% in male and 43.8% in female U. americanus, using a modified agglutination test (1∶25 titer). Seroprevalence differences between sexes were not significant (P > 0.05). Results of the present study add supportive data to the growing body of evidence suggesting that U. americanus has one of the highest T. gondii seroprevalences among all known intermediate hosts. In addition, our data emphasize the importance of understanding parasitic disease dynamics from a conservation perspective.

Prevalence of Toxoplasma gondii infection in pregnant women in Grenada is considered high. Little is known of the epidemiology of T. gondii infection in Caribbean Islands. Serum samples of 750 food animals in Grenada and Carriacou were tested for antibodies to T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT, 1∶25 or higher) were found in 23.1% of 247 pigs, 44.1% of 204 sheep, 42.8% of 180 goats, and 8.4% of 119 cattle. Seroprevalence increased with age, indicating postnatal acquisition of T. gondii. Antibody titers of 1∶200 or higher were present in 65 of 90 seropositive sheep, 61 of 77 seropositive goats, and 23 of 57 seropositive pigs. However, none of the cattle had a MAT titer of 1∶200, suggesting that bovines are a poor host for T. gondii. Results indicate that pigs, sheep, and goats could be important sources of T. gondii infection if their meat is consumed undercooked.

In the Peruvian Amazon, the white-lipped peccary (Tayassu pecari) is a desirable game species and is important for the local rural economy. Blood samples from 101 white-lipped peccaries from Peru were collected from 3 different conservation areas located in the municipalities of Manu and Tambopata, southeastern region of the Peruvian Amazon. Antibodies were assayed using the modified agglutination test (MAT, cut of value of 25). Antibodies to Toxoplasma gondii were found in 89.1% (90 of 101) of animals, with titers of 1 ∶ 25 in 9, 1 ∶ 50 in 25, 1 ∶ 100 in 20, 1 ∶ 200 in 14, 1 ∶ 400 in 12, 1 ∶ 800 in 9, and 1 ∶ 3,200 in 1; 87.7% and 89.2% of males and females, respectively, tested positively, and no association (P ≥ 0.05) with gender and occurrence of antibodies was observed.

The water buffalo (Bubalus bubalus) is important to the economy of several countries, especially in Asia and Brazil. Little is known of the impact of coccidiosis in buffaloes. Cattle and buffaloes are considered to have common species of Eimeria but critical cross transmissions have not been made because it is difficult to raise these hosts coccidian free. Clinical coccidiosis was confirmed post mortem in a 22-day old buffalo calf that died after a 3-4 day illness. Oocysts morphologically identical to Eimeria bareillyi were found in the feces and in sections of small intestine. Oocysts were often pyriform, sometimes with asymmetrical sides. The shorter end was flattened and approximately 5-6 microm wide. Unsporulated oocysts in feces were 23.2-29.5 x 16.5-22 microm in size with an average of 27.2 x 19.3 microm . Schizonts, gamonts, and oocysts were identified in sections of small intestine and they were located in entrocytes of jejunum and ileum. No coccidian stages were seen in sections of colon. This is one of the first confirmed cases of clinical coccidiosis in water buffalo.

Serum samples from 419 polar bears (Ursus maritimus) from Svalbard and the Barents Sea (collected 1990-2000) and 108 polar bears from East Greenland (collected 1999-2004) were assayed for antibodies against Toxoplasma gondii using the modified agglutination test. Antibody prevalences were 3.6% among cubs dependent on their mothers and 21.4% among subadults and adults. Among subadults and adults there was an interaction between population and sex, with similar prevalences among females (Svalbard = 19.5%, Greenland = 18.0%), but a high frequency among Svalbard males (28.7%) as compared to Greenland males (5.8%). The pattern was also significant after correcting for differences in age distribution. The sex-population interaction term is believed to be connected to area- and sex-specific feeding ecology. The prevalences of antibodies against T. gondii in Svalbard and Greenland were high compared to previously reported findings in polar bears from Russian and Alaskan areas.

The common brushtail possum (Trichosurus vulpecula) has well adapted to increasing urbanization, resulting in greater interaction with humans and their domestic pets. Wildlife species in urban areas face a higher risk of exposure to zoonotic pathogens and may be affected by parasites hosted by cats (Toxoplasma gondii) or dogs (Neospora caninum), yet it is unknown to what extent urban T. vulpecula are exposed to these parasites. Antibodies to T. gondii and N. caninum were assayed in sera of 142 adult possums from the city of Sydney, Australia. Using the modified agglutination test, antibodies to T. gondii were found in 9 (6.3%) of the 142 animals in titers of 1:25 (4), 1:50 (1), 1:100 (1), 1:800 (1), 1:3,200 (1), 1:6,400 (1), and 1:12,800 (1). Of some T. vulpecula multiple sera samples within a 2-yr frame could be collected, but seropositive animals in general were not recaptured after initial seroconversion. One possum had a high T. gondii titer on 2 consecutive bleedings, 14 mo apart, and seropositive possums appeared normal when captured. Sex seemed not to have an affect on antibody prevalence, but age and location may play a role. Antibodies to N. caninum were not detected in 1:25 dilution of sera in the N. caninum agglutination test, indicating that T. vulpecula may not have been exposed to this parasite. This is the first serological survey for T. gondii and N. caninum infections in urban T. vulpecula.

A 3-yr-old secundiparous female ring-tailed lemur presented to the Auburn University Small Animal Clinic with signs of dyspnea, lethargy, and anorexia. The animal died before she could be examined, and a full necropsy was immediately performed. Provisional necropsy findings included moderate pneumonia and hepatopathy. Acute interstitial pneumonia and focal hepatocellular necrosis were confirmed histologically. Lung impression smears, histopathology, electron microscopy, immunohistochemistry, and tissue culture isolation resulted in a diagnosis of acute disseminated Toxoplasma gondii infection, which was confirmed by polymerase chain reaction. The isolate of T. gondii was avirulent for mice and was named AU Tg1 and genetically is type II. The source of the infection remains unclear, but speculation suggests contaminated fruit or blackbirds (Passeriformes: Icteridae) acting as transport hosts for oocysts from nondomestic felids and feral cats on the property.

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 (MATor = 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.

Toxoplasma gondii was isolated from brain or heart tissue from 15 southern sea otters (Enhydra lutris nereis) in cell cultures. These strains were used to infect mice that developed antibodies to T. gondii as detected in the modified direct agglutination test and had T. gondii tissue cysts in their brains at necropsy. Mouse brains containing tissue cysts from 4 of the strains were fed to 4 cats. Two of the cats excreted T. gondii oocysts in their feces that were infectious for mice. Molecular analyses of 13 strains indicated that they were all type II strains, but that they were genetically distinct from one another.

Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.

The North American opossum (Didelphis virginiana) is host to at least 3 species of Sarcocystis: Sarcocystisfalcatula, Sarcocystis neurona, and a recently recognized Sarcocystis sp. A new name, Sarcocystis speeri, is proposed for the third unnamed Sarcocystis. Immunodeficient mice are an experimental intermediate host for S. speeri. Sarcocystis speeri sporocysts are 12-15 x 8-10 microm in size, and its schizonts are found in many organs of mice. Sarcocysts of S. speeri are found in skeletal muscles and they are up to 5 mm long and filiform. By light microscopy, the sarcocyst wall is thin (1 microm thick); ultrastructurally, the cyst wall is up to 1.8 microm thick and has characteristic steeple-shaped villar protrusions surmounted by a spire. Sarcocystis speeri schizonts are morphologically and antigenically distinct from schizonts of S. neurona, and S. speeri sporocysts were not infective to budgerigars (Melopsittacus undulatus).

Hepatic sarcocystosis was diagnosed in a horse in association with refractory bacterial osteomyelitis and plasma cell tumor of the maxilla and hepatic salmonellosis. Gross lesions included pleural, pericardial, and peritoneal effusions, hepatomegaly, gastric ulceration, colonic edema, and proliferative tissues filling 2 maxillary dental alveoli. Histologically, liver was characterized by severe suppurative, necrotizing, periportal hepatitis, and severe periacinar necrosis. Hepatocytes frequently contained protozoal schizonts in various stages of development. In mature schizonts, merozoites were often arranged radially around a central residual body, consistent with asexual division by endopolygeny. Ultrastructural features of merozoites included an apical conoid and polar ring, anterior micronemes, central nuclei, and absence of rhoptries. These protozoa did not react to antisera raised against Neospora caninum, Sarcocystis neurona, Toxoplasma gondii, or Hammondia hammondi. The microscopic and ultrastructural characteristics and immunoreactivity of this organism are consistent with a Sarcocystis sp. other than S. neurona. This is the first report of Sarcocystis-associated hepatitis in a horse. The life cycle of this organism and source of infection are unknown.

A 3-yr field trial was conducted on 8 commercial swine farms in Illinois to determine the effectiveness of a feline Toxoplasma gondii vaccine in reducing the exposure of swine to T. gondii. A vaccine consisting of live bradyzoites of the mutant T-263 strain, capable of preventing oocyst shedding by cats, was used in this study. Each farm was visited 3 times in 1994, 3 times in 1995, and once in 1996. Cats were trapped and inoculated with the T-263 oral vaccine during 1994 and 1995. On each visit, the following samples were collected: blood from pigs, cats, and mice for detection of serum antibodies to T. gondii, feces from cats to detect oocysts, and heart and brain tissues from rodents to determine the presence of T. gondii tissue cysts. The modified agglutination test (MAT), with a positive titer set at the 1:25 dilution, was used to determine serum antibodies. At first capture, 72.6% (61/84) of juvenile cats and 32.6% (31/95) of adult cats had no detectable antibodies (seronegative), indicating no prior exposure to T. gondii when they received their first vaccine. Of these first-time seronegative cats, 58.1% (18/31) of adult and 45.9% (28/61) of juvenile cats were recaptured and received a second dose of vaccine. Changes in the prevalence of T. gondii infection were evaluated from the prevaccination (1992, 1993) to the postvaccination (1996) period. Eleven cats (5%) were detected shedding oocysts between 1994 and 1996, of which 10 (90.1%) shed during 1994. The last detection of oocyst shedding by cats was during the first farm visit in 1995. There was a significant decrease in T. gondii seroprevalence for finishing pigs (P0.05, Wilcoxon sign rank test). There was a positive correlation (Spearman's p = 1.0, P0.0001) between the change in prevalence in juvenile cats and the change in prevalence in finishing pigs. The seropositivity rate (MATor = 1:25) in mice among all farms decreased from 4% in 1992-1993 to 0% in 1996. The mean prevalence of T. gondii tissue cyst isolation for mice on all farms decreased from 1.1% in 1994, to 0.8% in 1995, and to 0.5% in 1996. The results of this study suggest that the reduced exposure of pigs to T. gondii was due to the administration of the T. gondii vaccine to cats.

Serum samples from 296 horses slaughtered for food in the United States were tested for antibodies to Neospora caninum by the Neospora-agglutination test (NAT). Antibodies were found in 69 (23.3%) horses with titers of 1:40 (19 horses), 1:80 (19 horses), 1:100 (3 horses), 1:200 (7 horses), 1:400 (4 horses), and 1:800 (17 horses). This is the first serologic survey for N. caninum antibodies in horses.

Infection and immunity to toxoplasmosis induced by the RH strain of Toxoplasma gondii was compared in Sprague-Dawley (SD) and Wistar rats and in outbred Swiss Webster mice. All rats injected with up to 1,000,000 RH-strain tachyzoites remained clinically normal, whereas mice injected with only 1 live tachyzoite died of acute toxoplasmosis. Rats could be infected with 1 tachyzoite of the RH strain as shown by antibody development and by bioassay in mice. However, after 8 days, RH-strain organisms were recovered only inconsistently from SD and Wistar rat brains. Contrary to a report of sterile immunity to T. gondii infection in rats after immunization with live RH tachyzoites, we found infection immunity after challenge with the VEG strain. Toxoplasma gondii tissue cysts of the VEG strain could be recovered from most SD and Wistar rats, first injected with live RH-strain tachyzoites and then challenged with oocysts of the VEG strain. Our RH strain, and probably many others, passed for 50+ yr as tachyzoites has lost not only the capacity to form oocysts, but also shows a marked reduction or absence of tissue cyst (bradyzoites) formation.

A retrospective survey of 760 raccoons (Procyon lotor) revealed 9 animals with sarcocysts of Sarcocystis kirkpatricki in their brains. Six of the raccoons also had the organisms in their skeletal muscles, and 1 had them in the heart muscle. No age or gender predisposition was seen. Seven of the raccoons had concurrent viral diseases (canine distemper or rabies), suggesting that concurrent viral infections in raccoons may facilitate infection of brain tissue with S. kirkpatricki.

Transmission electron microscopy was used to study the ultrastructure of schizogony of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus). Schizogony occurred exclusively by endopolygeny within endothelial cells of pulmonary capillaries, venules, and small veins. Early schizonts were elongate with a large nucleus and nucleolus, surrounded by a pellicle consisting of a plasmalemma and an inner single membrane, and contained most of the organelles and inclusion bodies found in merozoites of Sarcocystis species. As development proceeded, schizonts increased in size and conformed to the shapes of the pulmonary blood vessels. As micronemes, dense granules, the conoid, and subpellicular microtubules disappeared, there was an increase in the size and number of mitochondria, Golgi complexes, and Golgi adjuncts (apicoplasts). As the nucleus elongated, there was a progressive increase in the number of spindles located at various intervals along the nuclear envelope. Eventually, 2 merozoites formed internally immediately above each spindle. During endopolygeny, a portion of the nucleus was incorporated into each merozoite bud along with 1 or 2 Golgi adjuncts, a Golgi complex, mitochondria, endoplasmic reticulum, and ribosomes. During merozoite formation, micronemes appeared in close association with the Golgi complex and gradually increased in number. The pellicle invaginated around the merozoites so they budded at the schizont surface leaving behind a small, central residual body. Dense granules appeared after merozoites were completely formed. Schizonts were 24 x 6.8 microm and contained 24-96 merozoites. Merozoites were 5.1 x 1.8 microm and were found free in the pulmonary air passages and pulmonary capillaries and within nearly all cells of the lung except red blood cells.

Vertical transmission of Neospora caninum was evaluated in BALB/c mice using an N. caninum-specific polymerase chain reaction (PCR) assay as a means of detecting parasite transmission to offspring. BALB/c mice were infected with the NC-1 isolate of N. caninum during pregnancy (days 8-15 gestation). Transmission of parasite, detected by PCR, was determined in 2- to 23-day-old offspring. When dams were infected on days 13-15 of gestation, transfer of parasites was detected in only a proportion of the litter. Infection between days 8 and 12 of gestation resulted in a high frequency of parasite transmission; every offspring from all litters was infected. The tissue locations of parasites in pups of different ages were determined. In young pups (2- to 4-days-old), the predominant sites of infection were the lungs and the brain. In older pups (7- and 23-days-old) the predominant site of infection was the brain. This study shows that PCR may be useful for evaluation of candidate vaccines against horizontal N. caninum infection, vertical transmission, or both.

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

Two pregnant llamas (Lama glama) infected with Toxoplasma gondii and their offspring were evaluated clinically and serologically. Llama 1 was inoculated orally with 1,000 infective occysts of the P89 strain of T. gondii at 82 days of gestation (DOG). Llama 2 became naturally infected with T. gondii between 26 and 119 DOG. Both llamas remained clinically normal and delivered healthy offspring. Sera collected from both llamas during pregnancy and from their offspring before and after colostral ingestion were evaluated for antibodies to T. gondii by the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and the Sabin-Feldman dye test (DT). In llama 1, MAT antibody titers were1:20, 1:320, 1:1,280, 1:640, and 1:80 at 82, 97, 109, 132, and 152 DOG, respectively. The MAT titers in naturally infected llama 2 were1:32, 1:320-1:640, and 1:1,280 at 26, 119-200, and 346 DOG, respectively. In both llamas, antibody titers in the DT were of similar magnitude as the MAT, but titers in the LAT and IHAT were inconsistent. Antibodies to T. gondii were not detected in precolostral sera obtained from offspring of both llamas suggesting there was no fetal T. gondii infection.

Opossums (Didelphis virginiana) were found to be hosts for 3 species of Sarcocystis: Sarcocystis falcatula with an avian intermediate host, S. neurona with an undetermined intermediate host, and a third, unnamed, species. Sporocysts from the intestines of 2 opossums (nos. 26 and 47) were fed to budgerigars (Melopsittacus undulatus), nude mice, and gamma-interferon knockout (KO) mice. Sporocysts of S. falcatula were not infective to nude or KO mice. Sporocysts of S. neurona induced encephalitis in KO and nude mice; only schizonts and merozoites were found in tissues of mice, and they reacted with anti-S. neurona serum raised against the SN-2 isolate of S. neurona originally obtained from tissues of a paralyzed horse. All 3 species of Sarcocystis were present in opossum no. 47. Sarcocystis neurona was isolated in cell culture from this opossum. Sporocysts from opossum no. 47 were lethal to budgerigars, indicating S. falcatula infection. Only 1 species of Sarcocystis (the third species) was found in opossum no. 26; the sporocysts were infective to KO and nude mice. Schizonts and merozoites of this species were predominantly in the liver but were also found in other tissues; schizonts did not react with anti-S. neurona serum. Merozoites of the third species were ultrastructurally distinct from S. falcatula and S. neurona merozoites. Sarcocysts were found in leg muscles of 2 mice killed 50 and 54 days after they were fed sporocysts from opossum no. 26. These sarcocysts had steeple-shaped protrusions on the cyst wall and were distinct from sarcocysts of S. falcatula and any other species of Sarcocystis.

Serum samples from 143 hunter-killed black bears were collected during the 1996 and 1997 black bear hunting seasons in eastern North Carolina. All samples were tested for antibodies to Toxoplasma gondii by the modified agglutination test. Antibodies to T. gondii were present in 120 of 143 (84%) bears. Females had significantly higher titers than males (Wilcoxon rank sums test, P = 0.045), and titers increased with age (Jonckheere test, P = 0.01). Samples collected during 1996 (n = 79) were tested for antibodies to Trichinella spiralis by enzyme-linked immunosorbent assay. No samples were positive for antibodies to T. spiralis.

A Leishmania sp.-like organism was found in the skin of a naturally infected 8-mo-old Red Dane female calf (Bos taurus) from Zimbabwe. There were multiple alopecic nodules, particularly on the face and udder. The nodules were up to 5 cm in diameter and larger ones were ulcerated and hemorrhagic. Numerous Leishmania-like amastigotes were seen in the skin lesions. Ultrastructurally, the organisms were oval to elongated (2-2.5 microm long), had a nucleus, a kinetoplast, and a flagellum. To our knowledge, this is the first report of Leishmania-like organisms found in an animal from Zimbabwe.

Immunity to toxoplasmosis was investigated in outbred pigs after feeding Toxoplasma gondii oocysts inactivated by irradiation. In 2 experiments 23, 2-3-mo-old pigs were fed (12 pigs only once and 11 pigs twice) approximately 10(5) oocysts irradiated at 0.3 or 0.4 kGy of 137Cs and 11 pigs served as unvaccinated controls. Eleven to 13 wk later, 15 vaccinated pigs and 5 nonvaccinated pigs were challenged orally with a high dose (10(5) or 10(6)) of oocysts, and 7 vaccinated and 6 nonvaccinated pigs were challenged orally with a low dose (10(2) or 10(3)) of oocysts; 1 vaccinated pig was not challenged. All nonvaccinated pigs challenged with a high dose became ill starting 4-5 days postchallenge (p.c.). One pig died day 9 p.c., 1 died day 16 p.c., and 1 was killed day 10 p.c. because of weakness. All vaccinated pigs, nonvaccinated pigs challenged with a low dose, and the 1 vaccinated unchallenged pig remained clinically normal. Bioassay of pig tissues (tongue, diaphragm, brain) in mice indicated fewer tissue cysts in tissues of vaccinated pigs compared with unvaccinated pigs following challenge with live oocysts. However, vaccination with 1 or 2 doses of irradiated oocysts did not prevent formation of tissue cysts even in pigs challenged with 10(2) oocysts.

A Sarcocystis-like organism was associated with encephalitis and myocarditis in an ataxic, emaciated adult male turkey (Meleagris gallopavo) from Gilmer County, West Virginia. Protozoal schizonts and merozoites were associated with areas of inflammation and occasionally necrosis in both the heart and the brain. The organisms divided by endopolygeny and stained positively with anti-Sarcocystis cruzi serum in an immunohistochemical test.

To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in a potential food source of infection for humans, we analyzed 43 isolates of T. gondii that had been collected from pigs at an abattoir in Iowa. Parasites were harvested as in vitro-grown tachyzoites, and their genotypes were determined at the SAG1 and SAG2 loci. On the basis of the allele identified at the SAG2 locus, isolates were grouped into 1 of the 3 primary lineages. Type II strains were by far the most prevalent, accounting for 83.7% of the isolates. The type III genotype was identified in only 16.3% of the isolates. These prevalences differ significantly from a previous sampling of isolates from animals but are similar to the frequencies with which they occur in human disease cases. Similar to the previously characterized strain P89, strains P62 and P105 appeared to have recombinant genotypes. The type I genotype was not identified in the isolates from pigs although these strains have previously been shown to account for approximately 10-25% of toxoplasmosis cases in humans.

Transmission and scanning electron microscopy were used to study the ultrastructure of the oocysts, sporocysts, and sporozoites of the VEG strain of Toxoplasma gondii and to compare the ultrastructure of sporozoites with tachyzoites (from the peritoneum of mice) and bradyzoites (from brain tissue cysts in mice). Oocysts were surrounded by a thin veil of finely reticulate material. The oocyst wall consisted of 3 layers and contained a previously unknown disc-shaped micropyle that appeared as a depression in the oocyst wall. The sporocyst contained 4 sporozoites and a residuum of lipid and amylopectin granules. The sporocyst wall was 3-layered with the innermost layer consisting of 4 curved plates held together at sutures by an interposed strip. Exposure to excysting fluid caused the interposed strip to separate from the curved plates, which curled inward releasing the sporozoites. Sporozoites had a posteriorly located nucleus and all the organelles typical for coccidian zoites. Sporozoites, tachyzoites, and bradyzoites had similar numbers of rhoptries but differed in the numbers and sizes of micronemes, dense granules, amylopectin granules, and lipid bodies.

Cryptosporidial infection was diagnosed histologically in the small intestine of a big brown bat (Eptesicus fuscus) from Oregon. This is the first report of cryptosporidiosis in a bat.

Serum samples from 185 chickens (Gallus gallus) collected from the various slaughter markets in and around Madras City, India were examined for antibodies to Toxoplasma gondii using the modified agglutination test incorporating mercaptoethanol. Antibodies (or = 1:25) to T. gondii were found in 39.5% of sera. Antibody titers of individual sera (% in parentheses) were 1:25 (8.1%), 1:50 (10.8%), 1:100 (6.5%), 1:200(2.7%), 1:400 (4.3%), 1:800 (5.9%) 1:1,600 (0.5%), and 1:3,200 (0.5%).

Sarcocysts of an unidentified Sarcocystis species were found in sections of skeletal muscles of a black bear (Ursus americanus) from North Carolina. Two sarcocysts in a section measured 45 x 37.5 microm and 67.5 x 50 microm and had a thin (2 microm) sarcocyst wall. The villar protrusions on the cyst wall were up to 2 microm long and up to 0.7 microm wide. The bradyzoites were approximately 6 X 2.5 microm in size. This is the first report of muscular Sarcocystis in a bear.

One of 12 necropsied cougars (Felis concolor vancouverensis) from Vancouver Island, British Columbia, Canada, shed Toxoplasma gondii oocysts confirmed by mouse bioassay. Eleven of the 12 cougars (92%) had antibodies to T. gondii by the modified agglutination test with titers of1:25 (1 cougar), 1:50 (8 cougars), and 1:500 (3 cougars). One additional cougar fecal sample collected from the Victoria watershed environment also contained T. gondii oocysts. In 1995, the largest reported outbreak of human toxoplasmosis was linked to municipal drinking water in Victoria, British Columbia. This study supports the initial hypothesis at the time of the outbreak that not only domestic cats, but also cougars, pose a risk to Victoria's water supply.

Acute generalized sarcocystosis was diagnosed in a capercaillie (Tetrao urogallus) from Finland. Microscopic lesions were seen in the heart, lungs, spleen, liver, and brain. Protozoa were found in all organs, especially in the lungs and spleen. Only asexual stages were observed. The parasite divided by endopolygeny. Schizonts were usually 10 microm wide and up to 55 microm long. Merozoites are 3-4 microm long and 1.5-2.0 microm wide. Sarcocysts and sexual stages are unknown. The parasite was considered to be a species of Sarcocystis with an unknown life cycle. This is the first report of acute sarcocystosis in capercaillie from Finland.

Serum samples from 70 (33 aborting and 37 non-aborting) dairy cows from a herd in California were analyzed for Neospora caninum antibodies in different laboratories by various serologic assays including enzyme-linked immunosorbent assay (ELISA) with recombinant antigens (Nc4.1 and Nc14.1), kinetic ELISA, whole tachyzoite lysate ELISA, immunostimulating complex (iscom) ELISA, antigen capture competitive inhibition ELISA, and by the indirect fluorescent antibody test. Eighteen percent of pregnant cows in this herd had aborted within 2 mo of the index case. All 70 cows had antibodies to N. caninum by at least 1 of the tests. Antibody levels to N. caninum in aborting cows as a group were higher than in nonaborting cows. However, it was concluded that no serological test could be used to establish definitively that N. caninum caused the abortion in an individual cow.

This report describes the clinical, microscopic, and ultrastructural findings in mink with muscular sarcocystosis. Three 2-3-mo-old mink were killed because they were ill with signs of progressive neurological disease. One mink had variable numbers of sarcocysts in multiple skeletal muscles. Sarcocysts were up to 300 microm in long and 20 microm wide. Ultrastructurally, the sarcocyst wall had numerous elongated 1.7-2.0-microm x 250-nm villar protrusions (VP). The VP had microtubules and irregularly distanced minute undulations. Both metrocytes and bradyzoites were present in sarcocysts. The mink with sarcocysts in muscles also had nonsuppurative meningoencephalitis and meningomyelitis. Similar brain lesions were found in other 2 mink from the same farm, but sarcocysts were not observed in the skeletal muscle of these animals. This is the first report of muscular sarcocystosis in mink.

Sarcocystis levinei Dissanaike and Kan, 1978, is redescribed because the original description was a mixture of 2 species, the amended S. levinei and the newly recognized S. buffalonis Huong, Dubey, Nikkilä, and Uggla, 1997. In histological sections, S. levinei sarcocysts are microscopic, up to 640 microm long and up to 95 microm wide. Ultrastructurally, the cyst wall is thin (1.0 microm thick) with a minute undulating surface and smooth, hairlike villar protrusions arising at irregular distances from the cyst wall. The villar protrusions have a dome-shaped base (approximately 0.5 microm thick), a fingerlike middle part, and a tapering distal end (0.1 microm thick). The morphological features of the sarcocyst resemble those of S. cruzi (Hasselman, 1926) Wenyon, 1926, of cattle. Sarcocystis levinei sarcocysts were found in striated muscles including heart, esophagus, tongue, and skeletal muscle. The buffalo myocardium is parasitized exclusively by S. levinei, whereas1 Sarcocystis species may occur concurrently in other muscular tissues of water buffaloes. Two dogs, but not 2 cats, fed water buffalo hearts infected with S. levinei sarcocysts shed sporocysts measuring 9.5-10.5 x 14.0-16.5 microm starting from days 16 and 18, respectively.

The effect of storage of Toxoplasma gondii tissue cysts in various NaCl solutions at different temperatures was studied. Tissue cysts from rodent brains were suspended in 0.85%, 2.0%, 3.3%, and 6.0% aqueous NaCl solutions. After storage at 4-20 C for various time intervals, brains were bioassayed in mice for viability of T. gondii. At 4 C, tissue cysts survived for at least 56 days in 0.85% NaCl, for 49 days in 2.0% NaCl, and for 21 days in 3.3% NaCl solutions. At 10 C, tissue cysts survived for at least 21 days in 0.85%, 2.0%, and 3.3% NaCl solutions. At 15 C, tissue cysts survived for at least 21 days in 0.85% NaCl, 14 days in 2.0%, and 3.3% NaCl solutions. At 20 C, tissue cysts survived for 14 days in 0.85% NaCl, 7 days in 2.0% NaCl, and 3 days in 3.3% NaCl solutions. Tissue cysts generally did not survive in 6.0% NaCl solution at any temperature.

Serum samples from 1,264 feral pigs from Ossabaw Island, Georgia were initially screened for antibodies to Toxoplasma gondii by the modified agglutination test (MAT) using whole-formalinized tachyzoites and mercaptoethanol. Seropositive samples were also tested by the Sabin-Feldman dye test, the latex agglutination test (LAT), and the indirect hemagglutination test (IHAT). Ossabaw Island is a remote, barrier island located southeast of Savannah, Georgia. Antibodies to T. gondii were found in 11 (0.9%) of 1,264 pigs. The antibody titers were 1:20 (1 pig), 1:80 (2 pigs), 1:160 (2 pigs), 1:320 (4 pigs), and 1:640 (2 pigs) by the MAT, and 1:8 (2 pigs), 1:16 (3 pigs), 1:32 (1 pig), 1:64 (2 pigs), 1:128 (1 pig), andor = 1:256 (2 pigs) by the Sabin-Feldman dye test. By the LAT, 5 pigs had a titer ofor = 1:64 and by the IHAT all 11 pigs had a titer of1:64. Antibodies (MAT titer,or = 1:25) were found in 31 (18.2%) of 170 feral pigs from mainland Georgia. This seroprevalence on the mainland was significantly higher (P0.0001) as compared on Ossabaw Island. The markedly low prevalence of T. gondii on Ossabaw Island was attributed to the virtual absence of cats on the Island; only 1 domestic cat was known to be present.

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.

Videomicroscopy and transmission electron microscopy were used to study the interaction of Toxoplasma gondii sporozoites with cultured cardiopulmonary artery endothelial, embryonic bovine tracheal and Madin-Darby bovine kidney cells. No moving junction or exocytosis of rhoptries, micronemes, and dense granules was detected during the initial penetration of sporozoites into cultured cells, whereas constriction of the sporozoite and partial exocytosis of rhoptries occurred during movement of the sporozoite from the first parasitophorous vacuole (PV1) into the second vacuole (PV2). The PV1 was unusually large, lacked a tubulovesicular membrane network (TMN), and had an indistinct parasitophorous vacuolar membrane (PVM). Comparatively, the PV2 was small, had a distinct PVM, contained a well-developed TMN, and was surrounded by numerous host cell mitochondria. Sporozoites that passed completely through cells carried with them an envelope of host cell membranes and cytoplasm. Cultured cells occasionally endocytosed sporozoites that were enveloped by host cell material. After formation of the PV2, sporozoites replicated by endodyogeny to form tachyzoites.

Relapse is common in immunocompetent and immunosuppressed humans infected with Isospora belli and is believed to be associated with the presence of extraintestinal stages. In the present study, we examined this important stage in an AIDS patient using histological, immunohistological, histochemical, and ultrastructural methods to better understand the development and structure of this stage and to develop better means of detecting infections. Antisera made in rabbits to Isospora suis, Toxoplasma gondii, Hammondia hammondi, Sarcocystis neurona, Neospora caninum, and Caryospora bigenetica were tested against I. belli tissue cysts in the avidin-biotin peroxidase complex (ABC) immunohistological test. Most antisera reacted positively in the ABC test at dilutions of 1:100 but not at dilutions of 1:250. Some antisera to N. caninum and H. hammondi reacted positively at dilutions of 1:1,000 in the ABC test. Most reactive antisera stained the tissue cyst wall and not the enclosed zoite. Eight histochemical tests were examined and most were nonreactive with I. belli zoites or tissue cysts. Transmission electron microscopy revealed that the tissue cyst wall was composed of granular material and was directly beneath the parasitophorous vacuole membrane. Zoites were in the center of the tissue cysts and were surrounded by fibrillar material that appeared to originate from the zoite surface. Tubulelike structures were present in the granular tissue cyst wall and in the fibrillar material that surrounded the zoite. Zoites contained a crystalloid body. New findings in the present study consisted of identifying what are probably early tissue cysts that lack a developed tissue cyst wall, demonstrating that more than 1 tissue cyst can occupy a host cell, describing the distribution of micronemes and the shedding of zoite membranes, and identifying tubular structures in the inner tissue cyst wall and inner compartment.

Toxoplasma gondii-infected rats are considered important in the epidemiology of toxoplasmosis because they can serve as a source of infection for pigs and possibly for cats. To study the distribution of tissue cysts, 10 Sprague-Dawley female adult rats were fed 1 oocyst (3 rats, group A) or 10(5) oocysts (3 rats, group B) of the VEG strain or 10(4) oocysts of the GT-1 strain (4 rats, group C) of T. gondii. All rats in a group were killed at 1 time: 76 (group A), 240 (group B), and 443 (group C) days after oocyst inoculation (DAI). Tissue cysts were seen in the brains of all 10 rats by direct microscopic examination. Portions or whole organs from heart, lung, liver, spleen, small intestines, kidneys, skeletal muscle, eyes, mesenteric lymph nodes, stomach uterus, and tongue from all rats in a group were pooled by organ, digested in acid-pepsin solution for 60 min, washed in saline, and then bioassayed in mice. Based on bioassay in mice, tissue cysts were present in 3 extraneural tissues of rats from group A, 6 extraneural organs of group B, and in 10 extraneural organs of rats of group C. Tissue cysts were present in skeletal muscles and kidneys of all 3 groups. Thus, tissue cysts are formed both in neural and extraneural tissues of rats. Therefore portions of infected rats, excluding the head, can be a source of infection for pigs and cats.

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.