Mark L. Anderson

Neosporosis in cattle

During the past decade, Neospora caninum infection has emerged as an important reproductive disease in cattle throughout the world. Abortion, occurring during the middle of gestation, is the primary clinical sign of the infection in cattle. Surveys in several countries from three continents have identified N. caninum infection as the major diagnosed cause of bovine abortion. Both endemic and epidemic patterns of abortion may occur in herds. An important feature of this disease is that the protozoan parasite is maintained in cattle as a chronic infection which can be passed on to the fetus during pregnancy. Two methods for the transmission of the infection in cattle have been proposed and are the subject of current investigations. Horizontal transmission utilizes a two-host life cycle whereby the cow is infected from ingestion of coccidial oocyst stages shed by the definitive host. Experimental infections have confirmed that the dog is a definitive host for the parasite. There is epidemiological evidence that the dog has a role in the prevalence of the infection but, as yet, no confirmation that the dog is the source for natural infections in cattle. Vertical transplacental transmission of the infection is an important route of infection in many herds. Vertical transmission occurs because fetal infection frequently does not result in abortion but rather the fetus survives to be a persistently infected animal. A heifer calf that is born congenitally infected is capable of transmitting the infection to the next generation when she becomes pregnant, thus maintaining the infection in the herd. The clinical outcome of transplacental fetal infection with N. caninum is likely determined by maternal and fetal immune responses which involve humoral, and most importantly, cell-mediated immune factors. The diagnosis of the infection is assisted through histopathology and immunohistochemical examination of aborted fetuses and serologic testing of cattle for evidence of infection. Several types of serologic tests, based on the use of culture-derived organisms or recombinant N. caninum antigens are available. There are no proven control methods for the prevention or treatment of neosporosis. Suggested control measures focus on programs to reduce the number of congenitally infected animals retained in the herd and to minimize the opportunity for postnatal transmission from the environment.

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.

Canine Distemper Epizootic in Lions, Tigers, and Leopards in North America:

Canine distemper virus (CDV) infection occurred in captive leopards (Panthera pardus), tigers (Panthera tigris), lions (Panthera leo), and a jaguar (Panthera onca) in 1991 and 1992. An epizootic affected all 4 types of cats at the Wildlife Waystation, San Fernando, California, with 17 mortalities. CDV-infected raccoons were thought to be the source of infection in these cats. Two black leopards died at the Naibi Zoo, Coal Valley, Illinois, and 2 tigers died at the Shambala Preserve, Acton, California. Initial clinical signs were anorexia with gastrointestinal and/or respiratory disease followed by seizures. Canine distemper virus was isolated from 3 leopards, 3 tigers, and 3 lions that died or were euthanized when moribund. Monoclonal antibody testing identified the virus isolates as CDV. Gross and histopathologic findings were similar to those found in canids with distemper with a few exceptions. There were fewer lesions in the brain, and there was a pronounced type 2 cell proliferation in the lung, with inclusion bodies and CDV antigen demonstrated by immunohistology. Neutralizing antibody to CDV was found in high titers in serum from most animals but was absent or was found only in low titers in some cats that succumbed after CDV infection. There was a marked difference in neutralizing antibody titers when tests were done with different strains of CDV.

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.

Evidence suggesting a point source exposure in an outbreak of bovine abortion due to neosporosis

A Holstein dairy farm suffered an abortion outbreak due to neosporosis. Abortion losses were > 18%. Cows with the highest Neospora antibody titers were at the greatest risk of aborting. Mummified fetuses were found after the 43rd day of the outbreak. The epidemic curve was suggestive of a point source exposure, which is consistent with the hypothesis that Neospora can be spread by a definitive host.

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.

A bovine model of vaccine enhanced respiratory syncytial virus pathophysiology

A critical issue has been the observation that vaccination of children with a formalin-inactivated respiratory syncytial virus (RSV) vaccine is associated with disease enhancement. We have taken advantage of bovine RSV and our experience with this disease in calves to develop a natural model that parallels human disease. Using formalin-inactivated bovine RSV vaccine calves were either sham-vaccinated/infected, vaccinated/infected, or vaccinated/sham-infected and their clinical signs, pulmonary function, and histological lung lesions quantitatively scored. Interestingly there was significantly greater disease in vaccinated/infected calves and histological lesions in calves were similar to those of affected children. Finally, we note that vaccination did not induce neutralizing antibodies, but IgG antibodies were detected by ELISA. Our model of RSV enhanced disease is important because it provides quantifiable evidence of disease severity that can be applied to evaluate the mechanisms of immunopathology and the safety of candidate RSV vaccines.

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.

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

Systemic Neosporosis in a California Black-Tailed Deer (Odocoileus Hemionus Columbianus)

to Neospora infection develop a parasite-specific antibody response that may provide valuable information on the exposure status of the cow. At this point, however, neither fetal nor maternal serology is a satisfactory substitute for histologic examination of brain for the diagnosis of Neosporainduced abortion. Eleven cows aborted during this abortion storm. Neospora was confirmed in all 7 of the fetuses in which brain was available and suitable for examination. This report illustrates the emerging importance of this disease in areas other than the West Coast and Southwest. Because Neospora has been reported from every region of the United States, it should be included on the list of agents capable of causing abortion storms in dairy cattle irrespective of the geographic location of the herd.