Protection in a hamster model of congenital toxoplasmosis.

Almost uniform protection against congenital toxoplasmosis initiated by inoculations with cysts and oocysts of the parasite was seen in the hamster model, among strains of different genotypes. Because the RH immunization prior to pregnancy has to be controlled with medication for most of the hamsters to survive, and also some congenital transmission of Toxoplasma was observed during the chronic stage of the infection, the hamster is considered less practical than the rat and the BALB/c mouse models. It is concluded that the hamster model closely resembles protection against congenital infection in nature, where most of the pregnant women and ewes that experienced a toxoplasma infection previously, protect their fetuses against an infection with the parasite during pregnancy.

Toxoplasma gondii: prototype immunization of lambs against formation of muscle and brain cysts.

Nine lambs were immunized with 10(6) oocysts of the relatively avirulent ME-49 strain of Toxoplasma gondii, and were challenged 45 days later with 4 x 10(6) oocysts of the M3 strain of this parasite. Less than 5 tissue cysts of the M3 strain formed per 8 g of muscle - if any - judging from a bioassay in mice in which all mice survived. In contrast, mortality occurred in mice fed muscle specimens from 4 lambs that had not been immunized with ME-49 prior to challenge with the M3 strain. This finding suggests the possibility of creating a vaccine for lambs that could diminish or abolish lamb and mutton as sources of human infection with T. gondii, and which could also prevent toxoplasmosis abortion in sheep.

Toxoplasma gondii: congenital transmission in a hamster model.

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non-invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n=168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated. Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.

Toxoplasma gondii: an improved rat model of congenital infection.

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10(4) Toxoplasma gondii bradyzoites or 10(2) T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model.

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.

Toxoplasma gondii: cross-immunity against the enteric cycle.

Eight of nine cats inoculated with strain ME-49 and challenged with three different strains of Toxoplasma were immune to oocyst shedding, as ascertained with bioassays of their feces. In a second experiment, only toxoplasma asexual stages were seen in H&E stained gut sections of cats treated with suppressive doses of sulfamerazine and pyrimethamine starting 2 days after oral inoculation with cysts of the strain ME-49 and killed 6 days later. In a third experiment, four cats were similarly inoculated and treated for 20 days. Six weeks later, the cats received an oral homologous challenge with cysts, and none shed toxoplasma oocysts. An acceptable level of cross-protection was achieved with strain ME-49, and therefore, it can be used as a candidate strain from which antigens could be tested for enteric protection.

Toxoplasma gondii: differential protection rates by two strains against cyst formation in a rat model.

A previous infection with the ME-49 strain of Toxoplasma gondii (of low pathogenicity for mice), protected 17 of 20 rats against formation of brain cysts, following challenge with 10(3) oocysts of the high pathogenicity M3 strain, as determined by bioassay of rat brains in mice. The low pathogenic KSU strain did not afford comparable protection. Protection was further tested in rats that were orally or subcutaneously immunized with cysts or oocysts of the ME-49 strain, and later challenged with 2 x 10(2) cysts or 10(2) oocysts of the highly pathogenic strains M3, M-7741 and C. Protection ranged from 43 to 100%, compared to non immunized control rats and was independent of the stage of ME-49 strain and of the routes used to immunize the rats. The results obtained encourage further investigation into prevention of toxoplasmosis in humans and food animals.

Refinement of the mouse model of congenital toxoplasmosis.

The goals of the present investigation, focusing on the BALB/c mouse model of congenital toxoplasmosis, were: (1) to find a method to determine pregnancy in the mouse. The method has 100% sensitivity and 72% specificity; (2) to test congenital transmission during the chronic stage of toxoplasmosis. This occurred in 2 of 10 mice tested; (3) to investigate the relationship between the infective dose and the rate of congenital transmission. This was not demonstrated for doses of 10(2) to 10(3) bradyzoites and oocysts of Prugniaud, M3 and M7741 strains, with transmission rates of 3 of 8 to 6 of 10 mice inoculated; (4) to determine homologous and heterologous protection. Homologous protection was demonstrated with Prugniaud cysts, and heterologous protection was found between ME-49 and M3 cysts. This finding is consistent with the uniform natural protection against congenital toxoplasmosis seen in immune women and ewes.

Toxoplasma gondii: partial cross-protection among several strains of the parasite against congenital transmission in a rat model.

Rats were immunized with cysts of two Toxoplasma strains or with RH strain tachyzoites prior to pregnancy. The litters of the 13 rats that received homologous challenges with cysts during pregnancy, were all protected, whereas of 173 rats that received heterologous challenges with cysts or oocysts, only 21 protected their litters. 38.3 and 17% of rats immunized with the RH and with complete strains respectively, and 57% of control rats challenged with cysts, transmitted the infection congenitally. The percentages when similar groups were challenged with oocysts, were 33.3, 48.2, and 56.2%, respectively. Immunization with cysts did not completely protect against challenge with oocysts, even if the same strain was used. The divergence of these results from the complete protection against congenital toxoplasmosis observed in immune women and ewes, might be due to the use of excessive challenge doses in the model.

An investigation of sterile immunity against toxoplasmosis in rats.

The non-persistent BK strain was examined for its ability to induce sterile immunity in Wistar rats. Groups of 2-9 Wistar rats were inoculated subcutaneously with 5 x 10(4) BK strain tachyzoites per rat. Two months later, 46 rats were dosed by gavage with 2 x 10(1) cysts of the C, ME-49, Prugniaud, C-56, Elg, M-7741 or M3 strains. Another 26 rats were inoculated with 10(3) oocysts of the ME49, M7741, Bear or Hopa-Hopa strains of Toxoplasma gondii. After 2 months, the rats were euthanized and their brains screened microscopically for toxoplasma tissue cysts and bioassayed in mice if negative. As judged by bioassay, the BK strain of Toxoplasma induced statistically significant protection against reinfection only when rats were challenged with cysts of the C and Prugniaud strains or with oocysts of the ME49 strain. Nonetheless, cysts were detected microscopically only in 23% of brains of immunized rats challenged with oocysts of the Bear and Hopa-Hopa strains of Toxoplasma and none of those challenged with tissue cysts of any strain. Tissue cysts were detected in 43 and 48% of non-immunized control rats infected with tissue cysts and oocysts, respectively. The overall infection in control rats (microscopy and bioassay) was 70 and 72% for rats inoculated with cysts and oocysts, respectively. These results are consistent with the divergent results obtained by other investigators with regard to protection after challenge with different complete strains (cyst and oocysts forming) of the parasite, of rats immunized with incomplete strains.

Cyst burden in the brains of Wistar rats fed Toxoplasma oocysts.

Six strains of Toxoplasma oocysts were used to infect groups of 4-24 Wistar rats, with each rat being fed 10(1)-10(4) oocysts from a single strain. After 2 months, the rats were killed, their brains screened for Toxoplasma cysts and then bioassayed in mice if negative. Toxoplasma was either observed in the form of brain cysts or was recovered using the bioassay, from 113 out of 138 (82%) rat brains. As few as ten oocysts were capable of initiating a brain infection that lasted for at least 2 months in eight of the nine rats inoculated. However, judging from bioassay 10(2)-10(4) oocysts did not give rise to progressively higher rates of infection. Brain cysts were seen in only 68 of 138 rats (49%). The number of Toxoplasma cysts formed in the brains of rats was generally in the order of tens to hundreds. The frequency of infection in the brains with Toxoplasma and the number of brain cysts formed appeared to be influenced by the individual resistance of the rats as well as by the doses of oocysts and the Toxoplasma strains used. The information gathered is considered to be a basis for a rat model of immunity against acquired toxoplasmosis.

Fetal Toxoplasma infection after oocyst inoculation of pregnant rats.

Six groups totalling 53 Wistar rats were fed 10(4)oocysts from one of six different Toxoplasma strains at 15 days of pregnancy. The overall transplacental transmission rate was 51%. This varied between 10% and 80%, dependent on the strain used. The strains of Toxoplasma which are more pathogenic for mice were transmitted transplacentally more frequently than the strains of intermediate or low pathogenicity. There were no statistically significant differences in the rate of congenital transmission of Toxoplasmain rats fed oocysts (present work) or cysts (previous work).

Residual infection of 15 toxoplasma strains in the brain of rats fed cysts.

Thirty-seven groups of 4-32 Wistar rats were 2-10(3) cysts of 15 Toxoplasma strains. After 2 months, the rats were euthanized and their brains screened for Toxoplasma cysts and bioassayed in mice if negative. The brains of 323 of 411 rats (78.6%) were found to be infected 2 months after inoculation with Toxoplasma cysts. Two hundred cysts were necessary to infect nearly 90% of the rats. With lower doses, only 60% of the rats had residual brain infection. Brain cysts were formed only in 146 of 411 rats (35.5%). The numbers of cysts formed were in the order of tens to hundreds, only occasionally one or two thousands. The mean percentage of rats with brain cysts, and the number of cysts formed in rat brains by different inocula, increased with higher doses of cysts and then declined. This pattern is difficult to explain and similar results regarding the number of cysts formed have been published. In relation to the mean percentage of rats infected, there appears to be a plateau in infection with the higher inocula. Neither the number of rats with cysts in their brains nor the numbers of cysts formed were dependent on the Toxoplasma strain used, with the exception of one strain. Instead, individual variations were marked, and are presumably related to variations in the individual genetic resistance to Toxoplasma infection in the rat. The information gathered is considered a preliminary step for a rat model of immunity against acquired toxoplasmosis.

Some factors influencing transmission of toxoplasma in pregnant rats fed cysts.

An overall 44% transplacental transmission rate was observed in 221 rats fed cysts of 12 Toxoplasma strains at 15 days of pregnancy, with a range of 0-90% transmission. Considerable variability in the transmission rate was seen among different groups of rats that received similar Toxoplasma inocula; this is attributed to genetically based susceptibility to Toxoplasma among individuals of the outbred Wistar strain of rats. Transplacental transmission was more frequent in Long Evans than in Wistar rats. Significant differences in the rate of transmission were not found between rats that were fed similar Toxoplasma inocula 6-8 days or 15 days after conception. The frequency of transmission was not affected by the strain or dose of Toxoplasma used.

Serological responses of cattle after treatment and during natural re-infection with Fasciola hepatica, as measured with a dot-ELISA system.

The dot-ELISA reaction was used to study the dynamics of IgG titers in cattle naturally infected with Fasciola hepatica after anthelmintic treatment and during reinfection. Excretion/secretion products (ES) of the parasite were used as antigens for the dot-ELISA. IgG antibodies were no longer detectable by dot-ELISA, 4-6 months after nine animals received the first of three weekly doses of triclabendazole (15 mg kg(-1)) and were then maintained on a pasture free of F. hepatica metacercariae. Six fluke-free cattle began shedding F. hepatica eggs 3-6 months after grazing a pasture contaminated with metacercariae of the parasite. A detectable increase in dot-ELISA IgG antibody levels was observed 2-4 weeks after natural reinfection by grazing a similar pasture contaminated with F. hepatica metacercariae. The usefulness of the dot-ELISA system to diagnose chronic infection by serology is complicated by previous treatment against the parasite. It is concluded that the ES antigens can be useful to detect early infection of cattle with F. hepatica in a dot-ELISA system

Congenital transmission of experimental chronic toxoplasmosis in rats.

A 10% transplacental transmission rate was observed in litters from 89 Wistar rats chronically infected with Toxoplasmosis gondii, as judging from bioassays. The rats had been fed T. gondii 2 mo prior to mating. Six of 7 isolates of T. gondii were transplacentally transmitted. The frequency of transmission did not appear to be affected by the strain of T. gondii or the size of the inoculum.

The incidence and economic significance of ovine toxoplasmosis in Uruguay.

Antibodies to Toxoplasma gondii were measured before and after pregnancy in a 1:64 dilution of sera with the direct agglutination test in 1613 ewes from 18 farms in eight different counties of Uruguay from 1992 to 1994. The overall seroprevalence increased from 28.7% before mating to 38.5% after lambing in 2.5 years and thus the incidence was 9.8%. Losses due to toxoplasmosis during pregnancy were estimated to be 1.4-3.9% of the total number of ewes investigated, amounting to approximately US$1.4-4.7 million for the whole country.

The incidence and economic significance of ovine toxoplasmosis in Uruguay.

Antibodies to Toxoplasma gondii were measured before and after pregnancy in a 1:64 dilution of sera with the direct agglutination test in 1613 ewes from 18 farms in 8 different counties of Uruguay from 1992 to 1994. The overall seroprevalence increased from 28.7% before mating to 38.5% after lambing in 2.5 yr and thus, the incidence was 9.8%. Losses due to toxoplasmosis during pregnancy were estimated to be 1.4 to 3.9% of the total number of ewes investigated, amounting to approximately US$1.4-4.7 million for the whole country.

Separation of toxoplasma cysts from brain tissue and liberation of viable bradyzoites.

A method was developed to separate Toxoplasma gondii cysts from mouse brain tissue and to liberate intact bradyzoites from cysts. Brains were blended in a Waring blender with 20% dextran solution and the homogenate was centrifuged at 4,000 g for 10 min. Cysts present in the sediment were digested by adding an equal amount of a solution containing 1 g NaCl, 1.4 ml HCl, and 1 mg pepsin (1:60,000 assay activity) per 100 ml water, and incubated at 37 C for 1 min. This method is harmless for bradyzoites, as tested by bioassays in mice. It compares favorably with published methods for separating cysts that require 3 centrifugations to achieve similar results.

Sarcosporidian infection in pigs in Uruguay.

Examination of enzymatic digests of samples of the crux diaphragm obtained at abbatoirs in Montevideo, Uruguay, indicated that 57.2% of 269 pigs weighing 90-140 kg were infected with Sarcocystis sp. The morphology of sarcocystis in H & E stained sections indicated that they were Sarcocystis miescheriana.