Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ⁺CD8⁺IFN-γ⁺ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ⁺CD4⁺ T cells were also found to increase in the infected mice, however later than CD8⁺ T cells. Interestingly, splenic and MLN CD4⁺CD25⁺ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ⁺CD8⁺ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.     

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.     

Serological analysis of calves experimentally infected with Neospora caninum: a 1-year study

A serological study was conducted with calves experimentally infected with the protozoan parasite Neospora caninum. The animals were inoculated with either a low or high dose of N. caninum tachyzoites and temperature responses monitored daily for the first 2 weeks after inoculation. Blood samples were collected before inoculation, and at regular intervals thereafter for 1 year. Serological analysis was achieved using an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and an IgG avidity ELISA.Injection of Neospora produced a significant rise in rectal temperature in the high dose group. In addition, the lymph node draining the site of inoculation increased in size following injection in all animals, in both infected groups, before returning to normal by day 14 after injection. Both groups given N. caninum produced specific antibody that was detected by the IFAT and the ELISA, which remained elevated for the 12-month duration of the experiment. The specific Neospora antibodies produced did not cross-react in an IFAT for the detection of antibodies to Toxoplasma gondii. IgG avidity increased 2 weeks after inoculation, in both infected groups, until week 12 when infection was well established. There was a little difference between the two infected dose groups. This study demonstrates that the two different doses of N. caninum produced a similar antibody response, and that the higher dose also induced a febrile reaction. The IgG avidity ELISA was successful at distinguishing between recent and long-standing infection in this study. However, in both groups, there was fluctuation in the levels of specific antibody throughout the yearlong study, which accords with similar experiments in pregnant cattle, where it has been suggested that fluctuation may indicate periodic recrudescence of infection and a re-stimulation of antibody production by antigen.     

Udder health in dairy cattle infected with Neospora caninum

Blood samples were collected from 3449 cows on 57 representative Ontario dairy herds during the summer of 1998 and analysed for antibody to Neospora caninum using an ELISA. Forty-eight herds (2742 cattle) contained at least one N. caninum-seropositive animal. Two composite milk samples were collected from all cattle: the first on the day of blood collection and the second 68 to 365 days later. All milk samples were submitted for bacteriological culture. Ontario Dairy Herd Improvement Corporation (DHI) data were available for 3162 cattle in the 57 herds at the time of bleeding. Furthermore, complete DHI data were available for 1658 cattle that were culled between 12 and 24 months following blood collection. Using a standardised ELISA sample-to-positive (S/P) cut-off of ≥0.45, the corrected seroprevalence was 8.2% overall and 10.1% within seropositive herds. At blood collection the odds of N. caninum-seropositive cows having a high linear score (≥4.0; equivalent to a somatic cell count ≥200,000 cells/ml) was 27% less than for seronegative animals. Similarly, at the time of culling, the odds of having a high linear score was 22% less in N. caninum-seropositive cattle. Overall, linear score was lower in N. caninum-seropositive cattle at culling. After controlling for herd, parity, days in milk, and the interval between collection of milk samples, the odds of N. caninum-seropositive cattle testing positive for an environmental pathogen (i.e. environmental Streptococcus species and coliforms) on the second milk sample was 56% less than for seronegative animals. The odds were 83% less at a higher ELISA S/P cut-off of ≥0.70. Finally, the odds of N. caninum-seropositive cattle developing a new infection with a major pathogen (environmental or contagious) were 60% less than seronegative cows using the higher ELISA S/P cut-off.     

Immune response to Neospora caninum native antigens formulated with immune stimulating complexes in calves

The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups.     

Serological responses to Neospora caninum in experimentally and naturally infected water buffaloes (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important natural host for Neospora caninum. Serologic responses to N. caninum were studied in experimentally and naturally infected water buffaloes in Brazil. Antibodies were assayed by the indirect fluorescent antibody test (IFAT) using a cut off value of 1:25. Six buffaloes were each inoculated subcutaneously with 5 x 10(6) live culture-derived tachyzoites of the cattle Illinois strain of N. caninum, and two calves were kept as uninoculated controls. Post-inoculation (p.i.) blood samples were collected weekly for 8 weeks and then monthly until 1 year p.i. All inoculated buffaloes developed IFAT titers of 1:100 or more between 7 and 11 days p.i. and the titers remained elevated until 7 weeks p.i. Antibody titers peaked to 1:1600 in 1, 1:800 in 3 and 1:400 in 2, usually by 3 weeks p.i. Antibody titers declined to 1:25 or 1:50 in all the six buffaloes by 12 months p.i. IFAT titers to N. caninum remained at an undetectable level (< 1:25) in both control uninoculated buffaloes. To follow the dynamics of N. caninum antibodies, sera from 29 buffaloes and their calves were collected for 1 year and assayed for N. caninum antibodies; 23 of 29 calves were seropositive (IFAT of 1:100 or more) at 1-2 day of age. Of these 23 calves, 17 remained seropositive during the study, while six became seronegative at four (two calves), six (one calf) seven (two calves) and eight (one calf) months of age. These findings suggest a high rate of neonatal transmission of N. caninum in buffaloes.     

Survival, immune responses and tissue cyst production in outbred (Swiss white) and inbred (CBA/Ca) strains of mice experimentally infected with Neospora caninum tachyzoites

The present work compared inbred (CBA/Ca) and outbred (Swiss white) strains of mice for their capacity to cope with a Neospora caninum infection and to consistently produce tissue cysts. In each experiment Swiss white and CBA/Ca mice were given three different doses of NC-1 tachyzoites. Lymphoproliferative and humoral responses as well as cytokine production were evaluated eight weeks after infection (PI) whereas tissue cyst production and histopathology were assessed 4, 6 and 10 weeks PI in immunosuppressed mice. Tissue cysts were observed 10 weeks after infection only in CBA/Ca mice receiving the two highest inoculum doses. Furthermore this strain showed the highest specific lymphoproliferative response. A mixed cytokine response with elevated IFN-gamma and fairly low IL-4 and IL-10 secretion was recorded. In both strains, no lesions were observed in the tissues of infected mice. This study indicates that CBA/Ca female mice infected with 5 x 10(6) NC-1 tachyzoites represent a useful model for the study of specific maternal immune responses in pregnant animals.     

Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses

Different aspects of Neospora tachyzoite antigen recognition by Neospora-infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora-antigen recognition was studied in Neospora-infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy.Based on the frequency and intensity of recognition, four IDAs-17-18, 34-35, 37 and 60-62kDa antigens-have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17-18kDa antigen. We emphasize the important role that the 17-18kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.     

Treatment of canine pediatric Neospora caninum myositis following immunohistochemical identification of tachyzoites in muscle biopsies

A 7-week-old Irish wolfhound was evaluated for an abnormal hind limb gait. Quadriceps muscle atrophy was pronounced and patellar reflexes were absent bilaterally. Neospora caninum myositis was diagnosed by histopathologic and serologic examination and immunohistochemical staining of muscle. Substantial clinical improvement was noted after 18 weeks of treatment with clindamycin.     

Prevalence and transmission of Neospora caninum within infected Swedish dairy herds

In this long-term study, the changes in within-herd Neospora caninum seroprevalences and the relative importance of horizontal and vertical parasite transmission in Swedish dairy herds were investigated. The within-herd prevalences varied between 5.8% and 65.0% when the herds were first sampled. Comparing the prevalences of year 2000 with those found 2-3 years later, nine out of 14 investigated herds had a similar or lower within-herd prevalence while five herds had an increased portion of seropositive individuals. Three herds were free or almost free from infection at their last sampling. Changes in prevalence were not related to the level of prevalence that each herd had when first sampled. Antibody avidity measurements revealed that the majority of seropositive individuals in all herds were chronically infected. Dam-calf pair investigations and inspection of genealogic trees supplemented with antibody test results showed that vertical transmission was by far the dominant transmission route in all herds. Taken together, these results indicate that the prevalence of N. caninum infection in Swedish dairy cattle can be successfully reduced if actions to eliminate infection in a herd are combined with preventive measures to avoid re-infection.     

Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil

Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.     

Evaluation of vaccination with Neospora caninum protein for prevention of fetal loss associated with experimentally induced neosporosis in sheep

OBJECTIVE: To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing fetal loss associated with experimentally induced neosporosis in sheep. ANIMALS: 30 Dorset ewes. PROCEDURE: Ewes were randomly allocated to receive vaccination on days 1 and 60 of the study with a killed N caninum tachyzoite preparation in a commercially available adjuvant or a saline-adjuvant mixture. A ram was placed on pasture with the ewes from days 15 to 60. Blood was collected from ewes before primary and booster vaccinations and prior to experimental challenge with N caninum tachyzoite performed on day 90; sera were assessed via Neospora agglutination (NA) and immunofluorescence antibody (IFA) assays. Blood was collected from lambs before they suckled, and sera were tested for antibodies against N caninum. RESULTS: Of the 14 vaccinated ewes that became pregnant, 12 gave birth to live-born lambs; in contrast, 5 of 11 pregnant control ewes gave birth to live-born lambs. Whereas vaccination improved fetal survival in pregnant ewes challenged with N caninum tachyzoites, it did not appear to have any appreciable effect on transmission of N caninum to offspring, as indicated by results of NA and IFA assays. CONCLUSIONS AND CLINICAL RELEVANCE: The N caninum tachyzoite vaccine used in this study appeared to provide protection against fetal loss associated with experimentally induced neosporosis in a high proportion of pregnant ewes.     

Neospora caninum tachyzoites inoculated by the conjunctival route are not vertically transmitted in pregnant cattle: A descriptive study

The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5 × 10⁸ tachyzoites, group 3 contained two cows inoculated with 2.5 × 10⁸ tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG₁/IgG₂ ratios were ≤1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.     

C57BL/6 mice infected with Neospora caninum during administration of progesterone show bias toward type 2 immune response

To clarify the role of progesterone in the development of immune responses during pregnancy against Neospora caninum infection, C57BL/6 mice were given a progesterone pellet, and measured on Interferon-gamma and interleukin-4 production following the infection. IFN-gamma production in the prescribed group was significantly lower than that in the intact group on day 40 post administration. IL-4 producing cell population in the prescribed group was larger than that in the intact group. These results suggest that progesterone may alter the balance of cytokine production, and that the bias toward type 2 immune response may remain for a certain period after the infection.     

Footpad reaction induced by Neospora caninum tachyzoite extract in infected BALB/c mice

Little information is available regarding a delayed type hypersensitivity (DTH) reaction in neosporosis. In this study, we examined the elicitation of a DTH reaction in mice infected with Neospora caninum by inoculation of the footpad with tachyzoite antigens. The footpads of BALB/c mice infected with N. caninum and those of non-infected were injected with either the tachyzoite extract, or paraformaldehyde-fixed tachyzoites. In mice inoculated with N. caninum antigens on day 7 p.i. swelling peaked at 6h after injection of the tachyzoite extract. In mice inoculated on days 14, 28 and 56, swelling was observed between 6 and 72 h afterwards. Mice immunized with the tachyzoite extract plus adjuvant showed peak footpad swelling at 6h post injection, and the swelling had decreased at 24h or later. In contrast, mice injected before infection showed no specific swelling. In sections of footpads injected with the tachyzoite extract, exudate had accumulated at 6h post injection and clusters of infiltrated lymphocytes were observed at 48 h post injection. In mice administered anti-CD4+ cell monoclonal antibodies swelling had decreased at 24h post injection of the extract. These results indicate that mice infected with N. caninum produce a DTH reaction, which is a good indicator of the development of type 1 immune responses.     

Antibody response in goats experimentally infected with Toxoplasma gondii

Serum samples of five goats inoculated with Toxoplasma gondii were analyzed using the enzyme linked immunosorbent assay (ELISA), indirect hemagglutination (IHA) and western blotting (WB). Antibodies detected by ELISA peaked between 19 and 62 days after inoculation and persisted throughout the experiment with no association to parasitaemia. Using WB, the main antigens detected had molecular weights of approximately 68, 62, 50, 48, 42, 34, 28, 26, 22 and 19 kDa. Antibody titers of between 1:256 and 1:32000 were observed using IHA, with a significant drop in activity after treatment with 2-mercapto-ethanol between days 12 and 48. This coincided with the parasitaemic period that occurs between 5 and 64 days after inoculation.     

Teladorsagia circumcincta antibodies in serum and milk samples in experimentally infected lactating ewes

The aim of this study was to evaluate the IgG activity in serum and milk samples of experimentally infected lactating ewes with the nematode Teladorsagia circumcincta as well as its relationship to the degree of infection. In a previous study 28 pregnant ewes were divided into two homogeneous groups, with high (H) and low (L) level of parasitism, respectively. Blood and milk samples were taken weekly after lambing until the end of the lactation, 126 days post-partum (pp). IgG against T. circumcincta were measured by means of an indirect ELISA. The kinetic of the immunoglubulins in serum samples showed a very low activity at the beginning but gradually increased throughout the lactation; H group showed higher values most of the sampled days than L group. Contrary, IgG in milk samples remained high during the first month pp, then decreased around 38% and by the end of the study rose again. Antibodies in both samples were correlated and especially during the second month of lactation (r=0.3; p<0.001). With the aim to correlate the immune response and the degree of infection we found an inverse relationship (r=-0.2; p<0.05) during the second month of lactation between eggs and IgG in serum. However the correlation with immunoglobulins in milk was positive, mainly, on the last third of lactation (r=0.2; p<0.01). As a conclusion, the individual detection of total IgG antibodies against T. circumcincta in lactating ewes is highly dependent on the stage of lactation. Therefore, these associations should be confirmed under field conditions.     

Effect of feeding lasalocid to pregnant ewes experimentally infected with Toxoplasma gondii.

Sixteen 50 day gestational ewes were fed lasalocid at the rate of 30 g t-1 and were orally inoculated with 100 infective Toxoplasma gondii oocysts 5 days after beginning feeding of lasalocid. Seventeen control ewes were similarly inoculated with T. gondii and were not fed lasalocid. The rate of abortion and neonatal mortality in both treated and untreated ewes was similar, indicating that feeding lasalocid was not effective in preventing T. gondii abortion in sheep.     

Disseminated infection with Neospora caninum in a ten-year-old dog

Naturally-occurring neosporosis with multiple organ involvement was identified in a 10-year-old neutered male Basset hound dog. Clinical signs were first noticed 3 weeks prior to referral and consisted of crouched stance and mild pelvic limb ataxia. Dexamethasone administration had provided transient improvement. On presentation to the teaching hospital, clinical signs included depression, pelvic limb ataxia, inability to stand without assistance, and pain on deep palpation of the cervical and lumbar vertebral column. Lesions were found in the myocardium, liver, spleen, adrenal glands, brain, and spinal cord. Tachyzoites of Neospora caninum were found in the myocardium and adrenal glands. Organisms stained with anti-Neospora caninum, but not to anti-Toxoplasma gondii serum in an immunohistochemical test.