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Background In Brazil, studies on animals and humans in mainland areas have shown that most strains of Toxoplasma gondii are pathogenic to mice and exhibit great genetic variability. Results In this study, using a set of 11 PCR-RFLP and 15 microsatellite markers, we isolated and genetically characterised T. gondii strains from one cat and three rats on Fernando de Noronha Island. The cat had antibodies to T. gondii, which were revealed using a modified agglutination test (MAT, cut-off 1:25) and the seroprevalence among the 46 rodents was 15.2%. Viable T. gondii was isolated from one cat (TgCatBrFN1), two brown rats (TgRatnoBrFN1 and TgRatnoBrFN2) and one black rat (TgRatraBrFN1). Unlike the strains from mainland Brazil, these isolates were not pathogenic to outbred mice. The genotypes of these strains were compared with strains previously isolated on the island and in mainland Brazil. The analysis based on microsatellite data showed a limited genetic diversity of T. gondii on Fernando de Noronha Island with the majority of strains clustered into the following three groups: type II, III, and Caribbean 1. Conclusions There was little variation among strains within the same group, suggesting that the majority of strains circulating on Fernando de Noronha are derived from only a few strains that were recently introduced to the island, likely from imported cats. Except for the strain belonging to the Caribbean 1 group that originates from northeast Brazil, there was little evidence that strains from the other groups were introduced to Fernando de Noronha via mainland Brazil. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2150-4) contains supplementary material, which is available to authorized users.

Understanding the role of disease in population regulation is important to the conservation of wildlife. We evaluated the prevalence of Toxoplasma gondii exposure and Sarcocystis spp. infection in 46 road-killed and accidentally trapper-killed fisher (Martes pennanti) carcasses collected and stored at -20 C by the Pennsylvania Game Commission from February 2002 to October 2008. Blood samples were assayed for T. gondii antibodies using the modified agglutination test (MAT, 1 : 25) and an indirect immunofluorescent antibody test (IFAT, 1 : 128). For genetic analysis, DNA samples were extracted from thoracic and pelvic limb skeletal muscle from each carcass to test for Sarcocystis spp. using 18s-rRNA PCR primers. Antibodies to T. gondii were found in 100% (38 of 38) of the fishers tested by MAT and in 71% (32 of 45) of the fishers tested by IFAT. PCR analysis revealed that 83% (38 of 46) of the fishers were positive for Sarcocystis spp. Sequence analysis of 7 randomly chosen amplicons revealed the fisher sarcocysts had a 98.3% to 99.1% identity to several avian Sarcocystis spp. sequences in GenBank. Data from our study suggest that a high percentage of fishers in Pennsylvania have been exposed to T. gondii and are infected with Sarcocystis spp.

The protozoon Toxoplasma gondii has a worldwide distribution and affects many species of warm-blooded animals. In the Canadian prairies, mesocarnivores such as striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) have experienced an increase in density and distribution, and they are in close contact with human dwellings. However, there has been no systematic study on the seroprevalence of T. gondii in these mesocarnivore populations. The objectives of the current project were to determine the serum antibody prevalence of T. gondii in Canadian prairie mesocarnivores and to study the relationship between antibody prevalence and species, sex, age, location, and year of collection. Antibodies to T. gondii were found in 5 of 24 (20.8%) skunks from Saskatchewan trapped in 1999 and 5 of 40 (12.5%) in 2000. Seroprevalences for T. gondii in raccoons and skunks trapped in Manitoba were 2 of 10 (20%) raccoons trapped in 2002, 7 of 44 (15.9%) trapped in 2003, and 16 of 37 (43.2%) trapped in 2004; and in 13 of 99 (13.1%) skunks trapped in 2003, 29 of 131 (22.1%) trapped in 2004, 53 of 165 (32.1%) trapped in 2005, and 30 of 51 (58.8%) trapped in 2006. Age, location, and year, but not the host species, were important variables in the determining the seroprevalence of T. gondii in skunks and raccoons. Results confirm that T. gondii is endemic in the skunk and raccoon populations in the Canadian prairies.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 98 free-range chickens (Gallus domesticus) from Nicragua was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 84 (85.7%) of 98 chickens with titers of 1:5 in 10, 1:10 in eight, 1:20 in seven, 1:40 in nine, 1:80 in 11, 1:160 in one, 1:200 in 27, 1:400 in six, 1:800 four, and 1:3200 in one bird. Hearts and brains of 32 chickens with titers of 1:10 or less were pooled and fed to three T. gondii-free cats. Hearts and brains of 66 chickens with titers of 1:20 or higher were bioassayed in mice. Feces of cats were examined for oocysts. The cat fed tissues from eight chickens with titers of 1:10 shed T. gondii oocysts. The two cats fed tissues of 24 chickens with titers of 1:5 or less did not shed oocysts. T. gondii was isolated by bioassay in mice from 47 chickens with MAT titers of 1:20 or higher. All infected mice from six isolates died of toxoplasmosis. Overall, 41 of 170 (24.1%) mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 48 isolates (47 from mice and 1 from pooled tissues) using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed eight genotypes. Six isolates had Type I alleles, three isolate had Type II alleles and six isolates had Type III alleles at all loci. Four isolates had mixed infections. Two isolates have a unique allele at SAG1 locus and combination of I and III alleles at other loci. The rest 27 isolates contained the combination of Type I and III alleles and were divided into four genotypes. More than one genotypes were often isolated in chickens from the same household, indicating multiple genotypes were circulating in the same environment. This may explain the high frequency of mixed infections observed. High rate of mixed infection in intermediate hosts such as chickens may facilitate genetic exchange between different parasite lineages in definitive feline hosts. This is the first report of genetic characterization of T. gondii isolates from Nicragua, Central America.

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Serum samples from 315 horses from Costa Rica, Central America, were examined for the presence of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii by using the surface antigen (SAG) SnSAG2 enzyme-linked immunosorbent assay (ELISA), the NhSAG1 ELISA, and the modified agglutination test, respectively. Anti-S. neurona antibodies were found in 42.2% of the horses by using the SnSAG2 ELISA. Anti-Neospora spp. antibodies were found in only 3.5% of the horses by using the NhSAG1 ELISA, and only 1 of these horses was confirmed seropositive by Western blot. Antibodies to T. gondii were found in 34.0% of the horses tested, which is higher than in previous reports from North and South America. The finding of anti-S. neurona antibodies in horses from geographical areas where Didelphis marsupialis has wide distribution suggests that D. marsupialis is a potential definitive host for this parasite and a source of infection for these horses. Se examinaron muestras de suero de 315 caballos de Costa Rica América Central, se examinaron para detectar la presencia de anticuerpos contra Sarcocystis neurona, Neospora spp. y Toxoplasma gondii mediante la prueba de inmunoabsorción superficie (SAG) SnSAG2 mediante el ensayo inmunoenzimático (ELISA), el ELISA NhSAG1 y la prueba de aglutinación modificada, respectivamente. Se encontraron anticuerpos anti-S. neurona en el 42,2% de los caballos mediante la prueba SnSAG2 ELISA. Los anticuerpos anti-Neospora spp. sólo se encontraron en el 3,5% de los caballos mediante la prueba ELISA NhSAG1, y sólo 1 de estos caballos fue seropositivo por Western blot. Los anticuerpos contra T. gondii fueron en el 34,0% de los caballos examinados, lo que es más elevado que en los informes anteriores de América del Norte y del Sur. El hallazgo de anticuerpos anti-S. neurona en caballos de zonas geográficas en las que Didelphis marsupialis tiene una amplia distribución sugiere que D. marsupialis es un potencial huésped de este parásito y una fuente de infección para estos caballos. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

The aim of this study was to identify risk factors associated with prevalence of Toxoplasma gondii infection in captive capuchin monkeys at a facility in the northeastern Brazil. Serum samples from 116 bearded capuchin (Sapajus libidinosus), nine blonde capuchin (Sapajus flavius), five black-capped capuchin (Sapajus apella), and four capuchin monkeys (Sapajus spp.) were tested for T. gondii antibodies using the modified agglutination test (MAT, cut-off ≥25); antibodies were found in 85.3% (99/116) of S. libidinosus, 55.6% (5/9) of S. flavius, 80.0% (4/5) of S. apella, and 75.0% (3/4) of S. spp. The risk factors associated with T. gondii seropositivity were ingestion of raw meat [OR = 4.13 (1.26; 13.50)] and old age [OR = 4.90 (1.70; 14.13)]. Results indicate a very high T. gondii seropositivity in these primate populations. To minimize exposure to T. gondii raw meat should not be fed to these animals.

Toxoplasma gondii tachyzoites were seen in the placenta of a late-term aborted Greenland muskox (Ovibos moschatus wardi) fetus in a captive herd at the San Francisco Zoo. The organism stained with anti-T. gondii polyclonal rabbit serum but not with anti-Neospora caninum serum. The dam had a Toxoplasma titer of > or =1:3,200 at the time of abortion and in each of the previous 3 yr (modified agglutination test). The muskox is a new host record for T. gondii.

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.

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.

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.

A species of Besnoitia from naturally infected rabbits from Argentina was propagated experimentally in mice, gerbils, rabbits, cats, and cell cultures. Cats fed tissue cysts from rabbits shed oocysts with a prepatent period of nine to 13 days. Sporulated oocysts were infective to gerbils, rabbits, outbred Swiss Webster and interferon gamma gene knockout mice. Bradyzoites were infective orally to gerbils and cats. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey kidney cells. Schizonts were seen in the lamina propria of the small intestine of cats fed tissue cysts; the largest ones measured 52 x 45 microm. Schizonts were also present in mesenteric lymph nodes, livers, and other extra-intestinal organs of cats fed tissue cysts. Oocysts were 10-14 x 10-13 microm in size. This rabbit-derived species of Besnoitia resembled B. darlingi of the North American opossum, Didelphis virginiana with an opossum-cat cycle, but it was not transmissible to D. virginiana, and B. darlingi of opossums was not transmissible to rabbits. Based on biological, serological, antigenic, and molecular differences between the rabbit and the opossum Besnoitia, a new name, B. oryctofelisi is proposed for the parasite from domestic rabbits from Argentina.

Fernando de Noronha is an archipelago of 21 islands and islets in the Atlantic Ocean, state of Pernambuco, Brazil, which has a varied biodiversity including alien species or sinantropic animals. The objective here was to determine the seroprevalence of Toxoplasma gondii in domestic and wild animals from Fernando de Noronha archipelago, Brazil. Between July 2007 and May 2010, blood samples were collected from 764 animals (533 domestic and 231 wild animals). Sera were tested by the indirect fluorescence antibody test (IFAT) or the modified agglutination test (MAT), or by both. Antibodies to T. gondii were found in 80 (80.0%) of 100 chickens ( Gallus domesticus ), 3 (3.0%) of 100 cattle ( Bos taurus ), 59 (60.8%) of 97 sheep ( Ovis aries ), 9 (81.8%) of 11 goats ( Capra hircus ), 7 (43.7%) of 16 horses ( Equus caballus ), 70 (59.3%) of 118 cats ( Felis catus ), 36 (39.6%) of 91 dogs ( Canis familiaris ), 13 (38.2%) of 34 black rats ( Rattus rattus ), and 157 (79.7%) of 197 cattle egrets ( Bubulcus ibis ). Results indicate endemic infection by this zoonotic parasite among the animal and avian fauna in this archipelago from Brazil.

Canine leishmaniasis and American trypanosomiasis (AT) are caused by related hemoflagellated parasites, Leishmania spp. and Trypanosoma cruzi, which share several common host species. Dogs are reservoirs for human infections by both pathogens. We determined the prevalence of antibodies to Leishmania spp. and T. cruzi in dogs from Grenada, West Indies. We examined 70 dog sera using the qualitative immunochromatographic dipstick tests (ICTs) based on recombinant antigens specific for visceral leishmaniasis and AT. Antibodies to visceral Leishmania were not detected in Grenadian dogs by ICT. Using the canine dipsticks for AT, antibodies to T. cruzi were determined in 3 (4.3%) of the 70 dogs. Results from this study indicate that dogs in Grenada are exposed in low levels to T. cruzi, but not to visceral Leishmania spp. at all.

The global distribution of leishmaniasis is rapidly expanding into new geographic regions. Dogs are the primary reservoir hosts for human visceral leishmaniasis caused by infection with Leishmania infantum. Natural infections with other Leishmania spp. can occur in dogs, but their role as reservoir hosts for other species of Leishmania is uncertain. Leishmania donovani is traditionally considered a visceralizing anthroponotic species; however, cutaneous leishmaniasis caused by L. donovani has been reported in Sri Lanka. In the present study, sera from 114 dogs in Sri Lanka were examined for antibodies to visceralizing Leishmania spp. Sera were tested by the canine immunochromatographic strip assays based on recombinant K39 antigen. Anti-Leishmania spp. antibodies were detectable in 1 of 114 (0.9%) dogs from Sri Lanka. Nonetheless, serological evidence suggests that leishmaniasis may be an emerging zoonosis in Sri Lanka.

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.

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.

To determine serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil.Prevalence survey.101 Thoroughbreds in Brazil.Blood samples were obtained from horses and tested for serum antibodies against S neurona by use of an immunoblot procedure with culture-derived S neurona merozoites as antigen, and for serum antibodies against T gondii and N caninum by use of a modified agglutination test with formalin-preserved tachyzoites and mercaptoethanol.Antibodies against S neurona and T gondii were detected in 36 and 16 of 101 horses, respectively. Cross-reactivity between antibodies against T gondii and S neurona was not detected. Antibodies against N caninum were not detected in any samples.The high prevalence of antibodies against S neurona detected in clinically normal horses emphasizes the importance of examining CSF for antibodies when establishing a diagnosis of equine protozoal myeloencephalitis.

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.

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.

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.

Dogs were investigated to determine if they are definitive hosts of Neospora caninum. Four dogs were fed N. caninum tissue cysts in infected mouse tissue, and two negative control dogs were fed uninfected mouse tissue. Dog faeces were examined daily for 30 days using a sucrose flotation technique. Three challenged dogs shed spherical to subspherical unsporulated oocysts, measuring 10 to 11 microns in diameter. Oocysts sporulated within 3 days and contained two sporocysts, each with four sporozoites. Outbred, inbred, and gamma-interferon knockout mice were inoculated with canine faecal extracts and monitored for evidence of neosporosis using a variety of morphologic, immunohistologic, serologic, and genetic analyses. Mice that received faeces from each dog observed to shed oocysts were demonstrated to have neosporosis by two or more techniques. One mouse was demonstrated to be infected with N. caninum by immunohistochemistry, ultrastructural analysis, and a species-specific PCR test. No evidence of neosporosis was observed in control animals. Based on this study, dogs are a definitive host of Neospora caninum.

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.

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%).

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.

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.

A nonsuppurative meningoencephalitis, previously presumed to be toxoplasmosis, was found in 53 capercaillies (Tetrao urogallus L.) examined at necropsy at the National Veterinary Institute, Uppsala, Sweden, between 1966 and 1985. Pronounced meningitis and encephalitis with perivascular cuffs of mononuclear inflammatory cells as well as focal gliosis were prominent histopathologic findings. Protozoa were frequently associated with these lesions. Ultrastructurally, the protozoa appeared to divide by endopolygeny, and merozoites had no rhoptries. Organisms from all 12 birds subjected to Sarcocystis cruzi immunohistochemical staining reacted positively but did not react to Toxoplasma gondii antiserum. The agent was, therefore, assigned to the family Sarcocystidae and was probably more closely related to species of the genus Sarcocystis than to T. gondii.

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.

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.

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.

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.