Emerging Infectious Diseases [Volume 5 No.2 / March - April 1999] Dispatches Neospora caninum Infection and Repeated Abortions in Humans Eskild Petersen,* Morten Lebech,* Lene Jensen,† Peter Lind,† Martin Rask,† Peter Bagger,‡ Camilla Björkman,§ and Arvid Uggla§ *Statens Serum Institut, Copenhagen, Denmark; †State Veterinary Laboratory, Copenhagen, Denmark; ‡Rigshospitalet, Copenhagen, Denmark; and §Swedish University of Agricultural Sciences, Uppsala, Sweden --------------------------------------------------------------------------- To determine whether Neospora caninum, a parasite known to cause repeated abortions and stillbirths in cattle, also causes repeated abortions in humans, we retrospectively examined serum samples of 76 women with a history of abortions for evidence of N. caninum infection. No antibodies to the parasite were detected by enzyme-linked immunosorbent assay, immunofluorescence assay, or Western blot. Neospora caninum, an intracellular protozoan parasite closely related to Toxoplasma gondii (1,2), was first described in dogs in Norway in 1984 and later in a wide range of other mammals including cattle, goats, horses, and sheep. The life cycle of N. caninum is only partially known, but the dog has recently been established as its definitive host (3). The pathogen's only known natural route of transmission (which can occur during sequential pregnancies in cattle) is transplacental (4). N. caninum is now recognized as the most common cause of repeated abortions and stillbirths in cattle, and infected herds have been reported in most parts of the world, including Scandinavia (4-6). Infected, live-borne offspring may have neurologic symptoms including progressive paralysis. When experimentally transferred to pregnant nonhuman primates, N. caninum has caused fetal infection. The fetal lesions closely resembled those in congenital toxoplasmosis (7). N. caninum organisms are morphologically very similar to T. gondii, the pathogen responsible for toxoplasmosis; however, the two species have distinct antigenic characteristics and can be distinguished by serologic and immunohistochemical methods (4). No case of N. caninum infection has been described in humans. However, because of the organism's close phylogenetic relationship to T. gondii and its wide range of potential hosts, the possibility of human N. caninum infection cannot be excluded. We investigated serologically the possible presence of N. caninum infection in Danish women who had repeated abortions of unknown cause. The Study The study included 76 women (mean age 30.8 years, range 19 to 41 years) who had had repeated abortions or intrauterine death of the fetus. Blood samples were obtained at the time of abortion or within 3 months of fetal death. The study participants had been referred to the Department of Gynecology and Obstetrics, Rigshospitalet, Copenhagen, Denmark, between 1 September 1991 and 31 October 1992 as part of a larger study of pregnant women with repeated primary or secondary abortions or repeated intrauterine fetal deaths. Serum specimens were tested for antibodies to N. caninum and T. gondii as described below. Findings The absorbence values for the human serum samples were 0.10 to 1.24 absorbence units, whereas the mean value for the presumed N. caninum-negative human control serum was 0.26 (0.13 to 0.56). The mean absorbence values for the high-positive and low-positive control pig sera were 1.73 (1.54 to 1.93) and 0.87 (0.85 to 1.07), respectively. As no true N. caninum-negative or -positive human sera were available, serum specimens with absorbencies 0.50 (n = 12) were selected for further investigation (Table). Table. Results of enzyme-linked None of the 12 specimens immunosorbent assay (ELISA)(sup a) tests for showed specific Neospora caninum and of the Sabin- fluorescence in the Feldman dye test(sup b) for Toxoplasma indirect fluorescence antibody test (IFAT) at gondii dilution 1:640 with N. ------------------------------------------------ caninum tachyzoites that Sample no. ELISA mean OD Dye test had been cultivated in vitro (6). (Sera had been ------------------------------------------------ diluted in twofold serial 90 0.720 0 dilutions from 1:20 in phosphate-buffered 107 0.627 0 saline.) Of the 12, only 262 0.578 1:50 3 had T. gondii 264 1.238 0 antibodies. The reactivities in the N. 276 1.043 1:50 caninum enzyme-linked 279 1.032 0 immunosorbent assay 282 0.647 1:10 (ELISA) were not associated with the 285 0.656 0 presence of T. gondii 287 1.143 1:6250 antibodies (Table). Only 1 of the 12 human serum 289 0.818 0 specimens tested showed 295 0.583 reactivity against the N. 297 0.541 0 caninum antigen by Western blot analysis. ------------------------------------------------ This specimen, number (sup a)The ELISA was modified from Björkman 279, recognized an et al. (8,9). A T. gondii-negative antigen with apparent human serum specimen was used as a molecular weight of 60 presumed N. caninum-negative control, kDa (Figure, lane 11 and and serum specimens from 12). This antigen was not experimentally N. caninum-infected recognized by the N. pigs were used as positive controls. A caninum-positive pig low-positive serum was collected from sera, and serum 279 did a pig infected 11 days before sampling not recognize any of the and a high-positive control serum was low-molecular weight pooled from pigs infected for at least antigens recognized by 3 weeks (10). No reaction in human the N. caninum-positive sera was definitely positive. pig sera. Three serum (sup b)The dye test was used to demonstrate specimens reacted with antibodies to T. gondii as described the T. gondii antigen; 11) but using in vitro cultured T. all were T. gondii. (gondii-positive in the dye test. Because of the biologic similarities between N. caninum and the human pathogen T. gondii, it has been speculated that N. caninum could be transmissible to humans. Since repeated abortions and stillbirths are common manifestations of neosporosis in cattle (4), women with a history of repeated abortions seemed an obvious category to investigate for human N. caninum infection. However, in this study of serum samples from women with repeated abortions, no evidence of N. caninum infection was detected. The assays we used were based on methods used for T. gondii analyses; we used the same conjugates and serum dilutions found optimal in these analyses. The N. caninum immunostimulating complex antigen has a high specificity (14) and has been used for serologic investigations in different animal species (8,9,15). It was therefore anticipated that it would be applicable in a human system as well. However, because we could not define a proper cut-off for the assay, we further investigated the serum samples with the highest ELISA absorbence values by IFAT, regarded as the reference test for N. caninum antibodies in different species (4), and Western blot. None of the human sera investigated showed any reactivity in IFAT. Only one of the specimens reacted with the N. caninum antigen in the Western blot. However, because it only reacted with a band not recognized by sera from the infected pigs, the reaction was considered unspecific, and cross-reactivity between T. gondii and N. caninum was not found. [fig] Figure. Western blot of Toxoplasma gondii or Neospora caninum antigen. Analysis was performed essentially as described by Sharma et al. (12) by using tachyzoites from in vitro culture of the N. caninum NC-1 isolate (13) and the T. gondii RH strain (10). Lanes 1-4 were probed with control sera and lanes 5-12 with human sera with high absorbencies in the N. caninum enzyme-linked immunosorbent assay. Lane 1: T. gondii-positive human serum and T. gondii antigen; Lane 2: N. caninum-positive pig serum and T. gondii antigen; Lane 3: T. gondii-positive human serum and N. caninum antigen; Lane 4: N. caninum-positive pig serum and N. caninum antigen; Lane 5 and 6: Serum 262: T. gondii and N. caninum antigens, respectively; Lane 7 and 8: Serum 264: T. gondii and N. caninum antigens, respectively; Lane 9 and 10: Serum 276: T. gondii and N. caninum antigens, respectively. Lane 11 and 12: Serum 279: T. gondii and N. caninum antigens, respectively. That we found no evidence of N. caninum infection in women who had repeated spontaneous abortions does not rule out the possibility that the infection might occur in humans. The predominant effects of neosporosis in dogs are primarily progressive neurologic signs including paralysis. It might, therefore, be worthwhile to examine human patients with clinical symptoms other than abortions, e.g., neurologic disorders of unknown etiology. Furthermore, the possible presence of N. caninum in patients with weakened immune systems should be considered. Researchers might continue the search for N. caninum by using serologic tests, as we did, or, alternatively, by using material collected at biopsy or autopsy for polymerase chain reaction or immunohistochemical analysis. --------------------------------------------------------------------------- Acknowledgments We thank Lisbeth Petersen, Lis Wassmann, and Ann Lene Andresen for skillful technical assistance. Dr. Petersen is a specialist in infectious diseases and tropical medicine at the Laboratory of Parasitology, Statens Serum Institut, Denmark's national reference center for diagnosis and research of human parasitic infections. His areas of expertise include immunology and epidemiology, primarily applied to malaria and congenital toxoplasmosis. Address for correspondence: Eskild Petersen, Laboratory of Parasitology, Statens Serum Institut, DK-2300 Copenhagen S. Denmark; fax:45-3268-3033; e-mail: ep@ssi.dk. References 1. Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 1988;192:1269-85. 2. Holmdahl OJM, Mattsson JG, Uggla A, Johansson K-E. The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences. FEMS Microbiol Lett 1994;119:187-92. 3. McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are definitive hosts of Neospora caninum. 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Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in cattle and water buffaloes in southern Vietnam. Vet Parasitol 1998;75:53-7. Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA URL: ftp://ftp.cdc.gov/pub/EID/vol5no2/ascii/petersen.txt Please note that figures and equations are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively. Greek symbols are spelled out. The following codes are used: (ft) for footnote; (sup) for superscript; (sub) for subscript; >/= for greater than or equal to.