Bradd C. Barr

Detection of Serum Antibody Responses in Cattle with Natural or Experimental Neospora Infections

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

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Neospora-like Protozoal Infections Associated with Bovine Abortions

Eighty bovine fetuses with presumed protozoal infections from a previous 2-year retrospective study were examined by immunohistochemistry using antisera against Neospora caninum. In 66 (83%) of the fetuses, protozoa were found that reacted positively with anti-N. caninum sera. In three (4%) additional fetuses, protozoa identified as Sarcocystis species did not react, and in two fetuses (3%) single protozoal clusters were found only in hematoxylin and cosin-stained slides. A group of 20 fetuses were chosen for further evaluation. They included 14 fetuses from the first group of 80 fetuses plus six additional fetuses that had large numbers of protozoa in the fetal brain. The 20 fetuses were examined immunohistochemically with antisera to N. caninum, Hammondia hammondi, and Toxoplasma gondii. Protozoa from 3/20 fetuses, identified as Sarcocystis species, failed to react with any antisera. In 16/20 fetuses protozoa reacted positively to antisera against N. caninum, and in most cases reacted to H. hammondi, and weakly to one or more of the antisera against T. gondii. Thickwalled protozoal tissue cysts were found in the brain of four of these 16 fetuses by transmissiosn electron microscopy. The cyst wall morphology was comparable to N. caninum. The results suggested that a single protozoal parasite of unknown identity was responsible for most of the bovine abortions. By immunohistochemistry, the unknown protozoon reacted most strongly and consistently to N. caninum antisera, but was antigenically distinct from N. caninum. Ultrastructurally, tissue cysts found in four fetuses most closely resembled Neospora caninum.

Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate

Studies were conducted to determine the pathogenic potential of the recently isolated bovine Neospora protozoa (BPA-1) for the bovine fetus. Cows chosen for study had Neospora titers < 160 using an indirect immunofluorescent antibody (IFA) test. Four experimental groups were studied. In group 1, 2 fetuses were inoculated in utero at 118 days gestation with culture-derived Neospora tachyzoites. A pregnant control cow was housed in the same pen, observed daily and screened serologically for evidence of exposure to Neospora. In group 2, 2 cows were infected with Neospora tachyzoites at 138 or 161 days gestation, and 1 control cow was given uninfected cell culture suspension simultaneously at 154 days gestation. Groups 3 (85 days gestation) and 4 (120 days gestation) each consisted of 2 cows infected with Neospora tachyzoites and 1 control cow given uninfected material at the same stage of gestation. Dead fetuses were surgically removed from the infected cows in group 1 on postinfection day (PID) 17. The histopathology was compatible with protozoal fetal infection, and protozoa were identified by immunohistochemistry. Viable fetuses were removed surgically from cows in group 2 on PID 28-30. The histopathology was compatible with protozoal fetal infection, protozoa were identified by immunoperoxidase techniques, and Neospora tachyzoites were reisolated in vitro from tissues of the 2 infected fetuses. In groups 3 and 4, the control fetus and 1 infected fetus were removed surgically between PID 26 and PID 33. The remaining infected cows were observed until fetal death or abortion occurred. In group 3, the fetus that was surgically removed from 1 infected cow had no pathologic abnormalities, and parasites were not found (PID 26). The second fetus in group 3 died in utero, and expulsion of a mummified fetus was induced on PID 67. Brain histopathology was compatible with protozoal infection, and parasites were identified by immunoperoxidase techniques. The fetus that was surgically removed (PID 32) from 1 infected cow in group 4 had lesions compatible with protozoal infection, and Neospora tachyzoites were reisolated in vitro from fetal tissues. The second infected cow in group 4 produced a full-term live calf that had a precolostral Neospora titer of 20,480. Clinically, this calf had depressed conscious proprioception in all limbs. Very mild lesions were found in the central nervous system, but protozoa were not found in the tissues. The results demonstrate that the bovine Neospora protozoa can be transplacentally transmitted, resulting in fetal infection and death, and mimics the naturally occurring fetal disease.

In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses

A Neospora sp. was isolated from the brains of two aborted bovine foetuses and grown continuously in vitro in bovine cell cultures. A comparison of the antigenic reactivity of in vitro cultivated tachyzoites with polyclonal antisera to Neospora caninum, Hammondia hammondi or Toxoplasma gondii revealed that the bovine protozoal isolates were similar to N. caninum and antigenically distinct from T. gondii. Tachyzoites of both bovine isolates had similar ultrastructural features, including an apical polar ring, conoid, electron-dense rhoptries and micronemes. The orientation of the micronemes, presence of micropores and a large number of electron-dense granules in the posterior portion of the bovine isolate tachyzoites differed from previous descriptions of N. caninum in vivo. Tachyzoites of the bovine isolates were ultrastructurally more similar to in vitro cultivated N. caninum tachyzoites than to tachyzoites of T. gondii or H. hammondi. The antigenic and ultrastructural similarities between N. caninum and the protozoal parasites isolated from aborted bovine foetuses in this study support the proposition that these parasites belong to the genus Neospora.

Neospora-Like Encephalomyelitis in a Calf: Pathology, Ultrastructure, and Immunoreactivity

Protozoal encephalomyelitis was diagnosed in a 3-day-old calf that was stunted, weak, and recumbent. Grossly, the calf had contracted tendons in the forelegs, a slightly domed skull, a porencephalic cyst in the cerebellum, ulcerative esophagitis, and abomasitis. Histologically, there was a multifocal nonsuppurative encephalomyelitis with clusters of protozoal tachyzoites and numerous protozoal cysts. The porencephalic cyst and gastrointestinal lesions appeared to be unrelated to the protozoal infection and were suggestive of a concurrent bovine virus diarrhea infection. A few groups of protozoal tachyzoites and numerous tissue cysts were found in neuropile, particularily in neurons of the spinal cord. By light microscopy, smaller tissue cysts were found in the brain (majority from 14 to 20 pm) than in the spinal cord (majority from 20 to 48 pm). The cyst walls ranged in thickness from < 1 pm to a maximum of 2 pm wide. Bradyzoites contained PAS-positive slender bradyzoites (5–8 × 1–2 μm). Tissue cysts reacted positively to anti-Neospora caninum sera; but unlike N. caninum, they were positive to 2 of 4 antisera against Toxoplasma gondii and to antisera to H. hammondi. Ultrastructurally, tissue cysts closely resembled a Neospora-like organism, including the finding of intemeuronal protozoal cysts, thick cyst walls, a lack of micropores in the bradyzoites, and the presence of numerous micronemes oriented perpendicular to the pellicle. Ultrastructural features in the calf protozoan that have not been reported for N. caninum in dogs included the presence of numerous tubulovesicular structures in the cyst ground substance and bradyzoite vesicles that contained small vesicular structures and short, flat membrane segments. The differences in antigenic reactivity and the minor morphologic differences between this bovine protozoan and N. caninum may reflect variations of a single organism dependent on factors such as the hosts species infected, the tissue infective stage studied, etc., or may indicate protozoal species/strain differences. The results also demonstrate antigenic cross-reactivity between this bovine apicomplexan, N. caninum, and T. gondii.

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis)

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.

Isolation and Characterization of Two Parasitic Protozoa from a Pacific Harbor Seal (Phoca Vitulina Richardsi) With Meningoencephalomyelitis

Two species of protozoans were isolated from a harbor seal with fatal meninogoencephalitis. Serologic reactivity was detected to both Sarcocystis neurona and Toxoplasma gondii. Parasites associated with brain inflammation and necrosis reacted only with immunohistochemical stains utilizing polyclonal antisera raised against Sarcocystis neurona. However, 2 distinct parasites were observed in cell cultures derived from the seals brain tissue. These parasites were separated by mouse passage and limiting dilution. Purified zoites from 1 isolate (HS1) reacted strongly with polyclonal antiserum to S. neurona and with the harbor seals own serum (1:2,560 for each) on indirect immunofluorescent antibody tests (IFAT), but weakly to antisera to T. gondii and Neospora caninum (1:40). Zoites from the second isolate (HS2) reacted positively with T. gondii polyclonal antiserum (1:81,920) and with the harbor seals own serum (1:640), but weakly to S. neurona and N. caninum antisera (1:80 or less). Amplification and sequence analysis of protozoal DNA encoding portions of the 18s ribosomal RNA (18s rDNA) and the adjacent first internal transcribed spacer (ITS1) were performed for both isolates, and resulting sequences were compared to those from similar protozoans. Based on molecular characterization, parasite morphology, serologic reactivity, histology, and immunohistochemistry, HS1 was indistinguishable from S. neurona, and HS2 was indistinguishable from T. gondii.

Neospora-Like Protozoal Infections Associated with Abortion in Goats

Neospora caninum was first identified and described in 1988 from a litter of puppies with encephalomyelitis. Since this first report, naturally occurring neonatal or fetal infections caused by Neospora-like protozoa have been described in cattle, sheep, and horses. Abortion or neonatal infection in cattle by this Neospora-like protozoa have been reported throughout the United States and in several other countries. Additionally, experimental infections have been produced in cats, rats, and mice. We report on 2 unrelated cases of abortion associated with a Neospora-like protozoan in pygmy goats. The first fetus and placenta (Fetus A), from a 2-year-old doe, were submitted in November 1989 from a small farm east of Sacramento, California. The doe had an uneventful pregnancy the previous year. The owner reported that abortions had occurred in 3 other does during the previous 2 years. These fetuses had been necropsied, but the etiology in each case was undetermined. No additional history on the flock was available. The fetus was 130 days gestational age and had a 24-cm crown-rump length. It was autolyzed, with blood staining of tissues and large amounts of deep red serous fluid in the body cavities. There were no significant gross pathologic findings. The placenta was very edematous. The second fetus and placenta (Fetus B) were submitted in March 1991 from a farm in the Sacramento Valley north of Sacramento, California. There was no history on this flock. The fetus had a 14-cm crown-rump length with no body hair. It was partially mummified with a small amount of fetal fluid in the thorax. There were no significant gross pathologic findings. Bacterial cultures of placenta, lung, liver, and abomasal fluid, darkfield examination of abomasal fluid, Chlamydial and viral cultures of pooled tissues, fluorescent antibody tests of kidney and placental impression smears for Leptospira spp. and Chlamydia psittaci, respectively, were performed as previously described and were all negative for both fetuses. Fluorescent antibody tests for border disease using frozen sections of liver and lung were negative in both fetuses. The total immunoglobulin G level in fluid from Fetus A was 2,720 mg/dl and in Fetus B was > 100 mg/dl. There was no antibody to Brucella ovis (enzyme-linked immunosorbent assay), bluetongue virus (agar-gel immunodiffusion), Leptospira spp. (microagglutination test), or Toxoplasma gondii (latex agglutination at a 1:16 dilution) in either fetus. Nitrate

Clinical, anatomic, and immunopathologic characterization of Babesia gibsoni infection in the domestic dog (Canis familiaris)

The pathology associated with acute, chronic, and recrudescent Babesia gibsoni infections was characterized in a group of 6 naturally or experimentally infected, spleen-intact and splenectomized dogs. All experimentally infected dogs became acutely parasitemic, lethargic, anemic, thrombocytopenic, and hemoglobinuric. Anatomic lesions associated, with the disease included diffuse nonsuppurative periportal and centrilobular hepatitis, multifocal necrotizing arteritis, membranoproliferative glomerulonephritis, reactive lymphadenopathy, diffuse erythrophagocytosis, and extramedullary hematopoiesis. The density of CD3+ lymphocytes within the liver sinusoids was markedly increased. Aggregates of large mononuclear cells with immunohistochemical features of activated macrophages were demonstrated in the central veins of the liver. Kupffer cells throughout the hepatic sinusoids appeared hypertrophic and prominent. The density of sinusoidal T lymphocytes, macrophages in central veins, and the degree of Kupffer cell hypertrophy were greatest in the splenectomized dogs. Multifocal deposits of IgM antibody were immunohistochemically demonstrated within the walls of inflamed arteries and renal glomeruli. The results of this study suggest that intense immunostimulation resulting in activation and expansion of T and B lymphocyte populations, macrophage recruitment and activation, vasculitis, glomerulonephritis and anemia contribute to the pathology associated with B. gibsoni infections.