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There are no reports of human infection with the protozoal parasite Neospora caninum, but it is possible that cases of neosporosis have been misdiagnosed as toxoplasmosis. Inoculation of pregnant monkeys with N. caninum results in transplacental transmission of the parasite and the induction of fetal encephalitis (3). N. caninum (Apicomplexa; Sarcocystidae; Toxoplasmatinae) was described and named in 1988 (6). It is closely related to Toxoplasma gondii as determined by ultrastructural and genetic comparisons (7), but its oocysts are shed by dogs instead of cats (10, 11). Infections are common in cattle, dogs, and a variety of other domestic and wild animals (7). Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that resemble toxoplasmosis both clinically and pathologically (7).

Humans infected with T. gondii are usually asymptomatic or suffer a flu-like illness, but the pathogen is clinically important in immunocompromised individuals and the fetuses of infected mothers (5). Infection in the immunocompromised host most commonly leads to encephalitis. Women who are first infected during pregnancy may miscarry or give birth to infants with encephalitis or hydrocephalus. Many prenatal infections are subclinical at birth but can lead to impaired vision or hearing, mental retardation, or convulsions (5).

The goal of this study was to determine if there is evidence of human exposure to N. caninum. Sera were collected from blood donors in central California, where N. caninum is recognized as a major cause of abortion in dairy cattle (1). The sera were screened for antibodies against N. caninum by an indirect fluorescent-antibody (IFA) test and immunoblotting. To confirm the specificity for N. caninum, the sera were similarly screened for antibodies against T. gondii.

Sera. A total of 1,029 serum samples were obtained from the Central California Blood Center (Fresno, Calif.). As a positive control, immune serum against N. caninum was generated by immunizing a rhesus macaque with formalin-killed N. caninum (NC-Liverpool strain) tachyzoites. This serum was negative by IFA testing for T. gondii antibodies. A human serum sample that was positive for T. gondii antibodies by IFA testing, but negative for N. caninum antibodies, was used as a positive control for the T. gondii screening.

IFA test. Human sera were screened for antibodies against both N. caninum and T. gondii by an IFA test. Slides spotted with whole N. caninum (NC-1 strain) tachyzoites were acquired from a commercial supplier (VMRD, Inc., Pullman, Wash.). For T. gondii testing, slides were spotted with whole tachyzoites prepared by scraping infected African green monkey kidney (Vero) epithelial cell (CCL 81; American Type Culture Collection) cultures and filtering the tachyzoites through a 3-µm-pore-size filter to remove Vero cell debris. Tachyzoites were then washed three times in phosphate-buffered saline (PBS; 25 mM NaPO₄-150 mM NaCl [pH 7.2]) and diluted to 10⁶ per ml. One drop of the solution was placed in each of 12 wells per slide and allowed to dry at 37°C. The cells were fixed with 80% acetone-20% methanol, rinsed with distilled water, and stored at 20°C until use.

Immunoblotting. N. caninum (NC-1 strain) and T. gondii tachyzoites harvested from infected Vero cells were used as antigens in immunoblots. Infected monolayers were removed by scraping, and cells were forced through a 20-gauge needle to release the tachyzoites. The organisms were washed twice with PBS, and the tachyzoites were purified from the Vero cell material by pelleting through a 30% Renografin (E. R. Squibb & Sons, Inc., Princeton, N.J.) pad at 58,424 × g for 30 min. Pellets were washed with PBS and suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer at approximately 10⁸ organisms per ml. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples through a 10% gel, proteins were electrophoretically transferred to Immobilon-P membrane (Millipore Corp., Bedford, Mass.), and the blots were blocked overnight in PBST (PBS plus 0.1% Tween 20) plus 2% bovine serum albumin (BSA-PBST). The blots were then incubated with human sera diluted 1:50 in BSA-PBST for 1 to 2 h at room temperature and washed three times with PBST for 10 min. Immunoblots were then incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated protein A (Pierce, Rockford, Ill.) for 1 h followed by three washes with PBST for 10 min. Horseradish peroxidase-conjugated protein A was detected by chemiluminescence with the SuperSignal CL substrate system (Pierce).

Results. Of the 1,029 human serum samples screened, 69 (6.7%) had titers of 1:100 in the N. caninum IFA test. The positive sera showed uniform peripheral membrane staining (Fig. 1A) or enhanced apical staining in addition to peripheral staining (Fig. 1B). Sera were considered negative for N. caninum antibodies if no staining of tachyzoites was observed or if peripheral staining was incomplete at a dilution of 1:100 (Fig. 1C). Incomplete peripheral staining is likely due to the reaction of antibodies not specific for N. caninum (13). All human sera were negative at a dilution of 1:200. To eliminate the possibility that a positive IFA test for N. caninum was the result of previous exposure to T. gondii, we tested the 69 N. caninum-positive sera against T. gondii. Fifty (72.5%) of the 69 samples positive for N. caninum antibodies by IFA testing were negative for T. gondii antibodies.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   IFA testing of human sera for reactivity against N. caninum. Slides were prepared with N. caninum tachyzoites as described in the text and treated with a 1:100 dilution of human sera. (A) Positive reactivity showing uniform peripheral staining of tachyzoites; (B) positive reactivity showing enhanced apical staining in addition to uniform peripheral staining of tachyzoites; (C) negative reactivity showing strong apical staining but no peripheral staining of tachyzoites.

View larger version (79K): [in this window] [in a new window]   FIG. 2.   Immunoblot analysis of reactivity of human sera against lysates of N. caninum (N), T. gondii (T), and Vero cells (V). The serum used to probe each blot is shown at the bottom. Strong bands of reactivity in the 35-kDa range are apparent with N. caninum lysates treated with either human serum sample. This reactivity is absent in lanes containing Vero cell or T. gondii lysates. Molecular masses are indicated in kilodaltons.

Discussion. In this report, we provide evidence for human exposure to, and possible infection with, N. caninum by using two different but complementary methods of serological testing. By IFA testing, 50 (4.9%) of 1,029 human sera were positive for N. caninum antibodies and negative for T. gondii antibodies, with titers of 1:100. Sixteen of these sera (1.6%) also reacted strongly with N. caninum antigens by immunoblotting. The reactive bands comigrated with those recognized by monkey anti-N. caninum serum. Moreover, antigenic bands of similar molecular mass were not observed when these sera were tested against T. gondii and Vero cell lysates.