Experimental porcine neosporosis

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Copvriaht 0 A P M I S 1998

APMIS 106: 475482. 1998 Printed in Denmark . All rights reserved


1PUUg ISSN 0903-4641

Exmimental porcine neoswrosis LENE JENSEN,' TIM K. JENSEN,' PETER LIND,' SVEND AAGE HENRIKSEN,' ARVID UGGLA2 and VIVI BILLE-HANSEN' 'Danish Veterinary Laboratory, Copenhagen, Denmark and 2National Veterinary Institute, Swedish University of Agricultural Sciences, Uppsala, Sweden

Jensen, L., Jensen, T. K., Lind, P., Henriksen, S. Aa., Uggla, A. & Bille-Hansen, V. Experimental porcine neosporosis. APMIS 106: 475482, 1998. Six gilts were inoculated intramuscularly with 2.5 X lo6 tachyzoites of Neospora caninum on three different days of gestation to study the pathogenic effect of Neospora infection in pigs, including possible transplacental transmission. The gilts were euthanized 59, 30, and 9/10 days postinoculation ( p i ) , corresponding to days 107, 102/106 and 110/111 of pregnancy. With the exception of one animal (euthanized day 9 p.i.) all gilts seroconverted as measured by the indirect, fluorescent antibody test (IFAT). Neosporosis with multifocal intralobular necrotizing hepatitis was seen in the two gilts inoculated 9/10 days before euthanasia. The uterus of one gilt inoculated 59 days before euthanasia revealed granulomatous and focal necrotizing endometritis with a corresponding multifocal necrosis of the trophoblasts of two fetuses. Transplacental neosporosis was indicated in the two fetuses by strongly elevated Neospora IFAT titres in pleural fluid and by the presence of multifocal necrotizing encephalitis and hepatitis together with non-suppurative myocarditis, pneumonitis, nephritis and hepatitis. Furthermore, N. caninum was re-isolated in cell culture from one of these fetuses. A third fetus from the same gilt revealed only disseminated, pinpoint necroses in the liver. Immunohistochemically, N. caninum tachyzoites were detected in association with histopathological changes in the liver and the endometrium of the gilts, and in the brain, liver, and allantochorion of the three fetuses. Key words: Neospora caninum; pigs; experimental; transplacental transmission; serology; pathology. Vivi Bille-Hansen, Danish Veterinary Laboratory, Bulowsvej 27, 1790 Copenhagen

Neospora caninum, a Toxoplasma-like tissue cyst-forming protozoon, is a common cause of bovine abortions and stillbirths worldwide, and sporadically the cause of congenital calf ataxia In other ruminants, such as goats and captive deer, N. caninum causes abortion and stillbirth, while congenital infection has been diagnosed in a lamb. Spontaneous neosporosis in monogastric animals has been reported in horses and dogs, in the latter of which the parasite was first described (Bjerkds et al. 1984; Dubey et al. 1988a). So far, a definitive host has not been

Received July 20, 1997. Accepted November 17, 1997.


identified. For review, see Dubey & Lindsay (1996). Experimentally, neosporosis with transplacental transmission of the parasite has been induced in pregnant cattle (Dubey et al. 1992), goats (Lindsay et al. 1995), sheep (McAllister et al. 1996), cats (Dubey & Lindsay 1989), dogs (Dubey & Lindsay 1990; Cole et al. 1995) and non-human primates (Barr et al. 1994). Fetal lesions of spontaneous and experimental neosporosis in these species are very similar and characterized by disseminated, non-suppurative necroses and non-suppurative inflammation in various organs, especially in the central nervous system (CNS), heart, and liver. In the present study, gilts in various stages of


JENSEN et al.

gestation were inoculated with N . caninum tachyzoites of bovine origin (Stenlund et al. 1997). The objective was to determine the pathogenic effect of the infection in pigs, including possible transplacental transmission.

MATERIALS A N D METHODS Animals and experimental design Six gilts, crossbreeds of Danish Landrace and Yorkshire, were obtained from an SPF herd free from infection with the porcine respiratory and reproduction syndrome virus (PRRSV). The gilts had been vaccinated against porcine parvovirus (PPV) and were seronegative in ELISA to Toxoplasma gondii (Lind et al. 1997). Pregnancies were confirmed by ultrasonography. Three groups, each of two gilts, were inoculated intramuscularly with 2.5X lo6 tachyzoites of the NcSweBl isolate, acquired recently from a stillborn calf and grown in Vero cell culture after isolation (StenIund et al. 1997). Inoculations were performed at days 48, 72/76, or 101 of gestation, respectively, and euthanasia at days 59, 30, and 9/10 postinoculation ( p i ) , corresponding to days 107, 102/106 and 110/111 of pregnancy, respectively. The animals were observed for clinical symptoms every day, and rectal temperature was measured during the initial 2 weeks. Each gilt and her fetuses were subjected to necropsy with the exception of two unopened fetuses, that were tested for PRRS virus at the Danish Veterinary Institute for Virus Research (DVIV), Lindholm, Kalvehave. Pathology At necropsy, the brain together with samples of cranial spinal cord, heart, lung, liver, spleen, kidney, ovary and uterus from the gilts was fixed in 10% neutral-buffered formalin. From the fetuses, the brain, samples of lung, heart, liver, spleen, kidney, and fetal membranes were fixed as above. Mummified fetuses were not necropsied. After proper fixation, tissue samples were embedded in paraffin, sectioned at 5 pm, and stained with haematoxylin and eosin (H&E). Selected sections were further specifically stained using a peroxidaseantiperoxidase immunohistochemical method (PAP), following standard operating procedure (SOP) employing a polyclonal rabbit antiserum against N. caninum. Microbiology Organ samples from the gilts were examined bacteriologically on indication. Two fetuses from each gilt were tested for Leptospira bratislava by IFAT, for PPV by cell culturing of pleural fluid and for antiPPV antibodies by ELISA, and they were subjected


to routine bacteriological examination. In the remaining fetuses, bacteriology was performed on indication; All examinations were performed at the Danish Veterinary Laboratory (DVL), following SOP Testing of fetuses for PRRSV by serology (antibodies in pleural fluid) and inoculation on cell cultures followed by immunoperoxidase assay took place at the DVIY Faecal samples collected twice a week during the first 3 weeks, and thereafter once a week, were examined using a modified McMaster technique for demonstration of helminth eggs and coccidial oocysts down to a size of 10 pm (Henriksen & Korsholm 1984). Additionally, smears were stained by using modified Ziehl-Neelsen technique to reveal oocysts of Cryptosporidium spp. (Henriksen & Pohlenz 1981).

Isolation of N. caninum in cell culture From each fetus, samples of medulla oblongata, brain and liver were pooled for examination by cell culture. Isolation of N. caninum was performed according to Conrad et al. (1993b). Briefly, the pooled samples were stored in sterile phosphate-buffered saline, pH 7.2 (PBS) with 1.3% dihydrostreptomycin and 1O6 IE/I penicillin, homogenized in PBS, and filtered through sterile gauze. Four ml of the suspension was mixed with 20 ml trypsin-EDTA 0.05% (trypsin 1:250, 01252-13-1 Difco, Detroit, USA; EDTA: titriplex 108418, Merck, Darmstadt, Germany) and incubated for 1 h at 37°C. After centrifugation, the pellet was resuspended and inoculated into tissue culture flasks (163371, Nunc, Roskilde, Denmark) with a monolayer of bovine turbinate (BT) cells (ECACC no. 89031603). The inoculated cells were grown in 10 ml Eagles minimal essential medium (MEM) with 10 mM HEPES and Glutamax- 1 (07290035E Gibco, Life Technologies, 4000 Roskilde, Denmark), 1.2 g/1 NaHC03, 0.13% dihydrostreptomycin, 100000 IE/l penicillin and 2-10% fetal calf serum (FCS) (Sera Lab., Crawley Down, UK). Upon detection, the parasites were transferred to Vero cells, grown in RPMI 1640 with Glutamax1 (71800-015, Gibco, Life Technologies), with 2.0 g/ 1 NaHC03, 0.13% dihydrostreptomycin, 100000 IE/1 penicillin and 1/2-1% FCS. Serology The Neospora isolate used as antigen, NC-I (Dubey et al. 1988b), was maintained in Vero cells, as described above, but using 2-5% normal horse serum instead of FCS. The parasites were harvested from the cell culture supernatant, filtered through a 3 pm polycarbonate filter, and washed twice in PBS. A parasite suspension of 5X lo7 per ml was used to coat teflon slides (PFTE 61.100.36, Boeterdael, Belgium), after which fixation in acetone took place for 5 mm. Blood samples were taken 1-3 times a week from the gilts. N . caninum-specific IgM and IgG antibodies


were assayed by indirect immunofluorescence test (IFAT) essentially as described by Conrad et al. (1993a). Plasma and fetal pleural or peritoneal fluid were tested from a dilution of 1:40 and l:lO, respectively, in twofold dilutions until the reactions were negative. Conjugates used were fluorescein-labelled goat anti-swine IgM(p) (02-14-03) and goat antiswine IgG(y) (02-14-02) from Kirkegaard & Perry Lab., Gaithersburg, MD, USA, diluted 1:40. Titres are given as reciprocal dilutions. The cut-off titres for seropositivity were 640 and 80 for gilt serum and fetal fluid, respectively.

RESULTS The main pathological and serological findings in the gilts and their fetuses are summarized in Tables 1 and 2, respectively. None of the six gilts had antibodies t o N. caninurn or T. gondii prior to inoculation. They were all asymptomatic, with normal temperatures (37.2-39.3”C) during the first 2 weeks p.i.

Gilt no.

At the time of euthanasia the number of fetuses, normally developed as well as mummified, was within the normal range for gilts. Fetuses tested for PPV, PRRS virus and pathogenic bacteria were all negative. Neither coccidian oocysts nor eggs of gastrointestinal helminths were detected in the faecal samples at any time. The gilts reached titres to N . caninurn of 1280 or higher in the IgM as well as the IgG class, with the exception of no. 5 (Fig. l), which showed only a slight rise in the IgM titre at day 9 p.i. (the day of euthanasia). Neosporosis was indicated in two fetuses (nos. 4 and 5 ) from gilt no. 1 by elevated levels of specific IgM and IgG antibodies in pleural fluid (Table 2). From fetus no. 4, the parasite was furthermore re-isolated in cell culture. At necropsy, disseminated pale pinpoint necroses were seen in the liver of fetus no. 3 from gilt no. 1, and in the liver of gilt no. 5. In gilt no. 1, the uterus segment containing fetuses nos.

TABLE 1. Gestation data, serological and pathologicaljndings in gilts inoculated with N. caninurn Day” of Day” of No.” of Maximum Detection of Histopathology inoculation euthanasia fetuses N. caninum N. caninum by antibodv titre immunoI ~ M I ~ G histochemistry








2 3 4 5

48 76 72 101

107 106 102 110

10/0 11/2 13/2 11/0

5120 1280 5120 80

20480 5120 5120 40












Granulomatous and multifocal necrotizing endometritis, mononuclear interstitial heDatitis nad nad nad Cuffings in cerebrum, focal necrotizing splenitis, focal mononuclear myocarditis, multifocal intralobular necrotizing hepatitis, mononuclear interstitial hepatitis Cuffings in cerebrum, focal mononuclear myocarditis, multifocal intralobular necrotizing hepatitis, mononuclear interstitial hepatitis

Day of pregnancy.

” No. of normally developed / mummified fetuses. “ Blood sampling was not performed after day 9. nad: No abnormalities detected.


JENSEN e l al.

4 and 5 revealed a dark hyperaemic, thickened and dry-appearing endometrium with similar lesions of the corresponding fetal membranes (allantochorion). By histopathology, the liver lesions were verified as multifocal intralobular necrotizing hepatitis with mononuclear interstitial infiltrations. Similar lesions were moreover evident microscopically in the liver of gilt no. 6 (Fig. 2). Additionally, gilts nos. 5 and 6 both revealed a few mononuclear cuffings in the cerebrum and focal mononuclear myocarditis, and gilt no. 5 a focal necrotizing splenitis. The uterus lesions of gilt no. 1 were characterized by granulomatous and focal necrotizing endometritis (Fig. 3 ) with multifocal necrosis of the trophoblasts of fetal membranes corresponding to fetuses nos. 4 and 5. These fetuses, moreover, showed multifocal necrotizing encephalitis (Fig. 5) and hepatitis together with non-suppurative myocarditis, pneumonitis, nephritis and hepatitis. By immunohistochemistry, N . caninum tachyzoites were detected sporadically in pathological foci in the liver of gilts nos. 5 and 6, in the endometrium of gilt no. 1 , as well as in the liver of fetus no. 3 from gilt no. 1. Furthermore, the parasite was identified in the brains of fetuses nos. 4 and 5, and finally in the fetal membranes of fetus no. 4 (Fig. 4).






The present study demonstrates that N . caninum infection is readily established in pigs after intramuscular injection of tachyzoites and that pig fetuses may be transplacentally infected. After inoculation, the infection rapidly be-

Fig. 2. Liver of Neospora-infected gilt no. 6, showing focal necrotizing hepatitis with mononuclear infiltration (H & E, X20). Fig. 3. Endometrium of Neospora-infected gilt no. 1 with granulomatous and focal necrotizing endometritis (H & E, X 10). Fig. 4. Trophoblast of fetus no. 4. N. caninum PAP staining of a cluster of tachyzoites ( X 100). Fig. 5. Necrosis in the brain of N. caninum-infected fetus no. 5 (H & E, X10).



TABLE 2. Serological and pathological findings in fetuses from gilt no. I Fetus no. N . canium detection IFAT-titre Cell culture ImmunoIgM IgG histochemistry 1,2, 6 and 7 3 4

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