Veterinary Parasitology 82 (1999) 327–333 Rapid communication Confirmation that the dog is a definitive host for Neospora caninum David S. Lindsay a,∗, J.P. Dubey b, Robert B. Duncan a a Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA b United States Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705, USA Received 5 February 1999; accepted 13 March 1999 Abstract Two mixed-breed littermate dogs were fed mouse brains containing tissue cysts of the NC-beef isolate of Neospora caninum. Both dogs excreted N. caninum oocysts in their feces. Dog 1 which was given methylprednisolone acetate (MPA) prior to ingesting tissue cysts, excreted oocysts on days 5 to 10 inclusive and on day 17 after ingesting tissue cysts. Dog 1 had a serum antibody titer of 1 : 200 in the indirect fluorescent antibody test (IFAT) 35 days after it was fed tissue cysts. Dog 2, which was not treated with MPA, excreted oocysts on Day 6 and Day 9 after ingesting tissue cysts. Antibodies to N. caninum were not found in a 1 : 25 dilution of serum on any examination period for Dog 2 during the study. Neospora caninum was not found in the tissues of either dog by histological or immunohistochemical means following necropsy 42 days after being fed tissue cysts. The identity of the oocysts excreted in the feces of the dogs was confirmed by mouse inoculation studies. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; Oocysts; Dog 1. Introduction Neospora caninum is an important cause of abortion in dairy cattle (Dubey and Lindsay, 1996). It had been postulated ever since its discovery that N. caninum had a coccidial ∗ Corresponding author. Tel.: +540-231-6302; fax: +540-231-3462; e-mail: [email protected] 0304-4017/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S0304-4017(99)00054-0 328 D.S. Lindsay et al. / Veterinary Parasitology 82 (1999) 327–333 life cycle that was similar to T. gondii (Dubey et al., 1988a, b; Dubey and Lindsay, 1996), but only recently have dogs been shown to excrete oocysts and be a definitive host (McAllister et al., 1998). The earliest that oocysts were seen in dog feces was 8 days after infection and they were excreted for 7–19 days. Oocysts extracted from the feces caused fatal infections in ␥-interferon knockout (␥-INF-KO) mice, and transmission electron microscopy and immunohistochemistry demonstrated that infection with N. caninum had been established. The effect of dog age, breed, and immune status on oocyst excretion is not presently known. Similarly, although the role of N. caninum oocysts in the epidemiology of neosporo- sis is unknown, it is likely that the oocyst plays an important role in the transmission of the parasite to cattle. Because of this, we conducted the present study to confirm and extend the findings of McAllister et al. (1998). Additionally, we report new findings on the biology of N. caninum oocysts. 2. Materials and methods 2.1. Inoculation and examination of dogs Two mixed-breed littermate dogs (Dog 1, female; Dog 2, male) about 14 weeks of age were used. Before use, both dogs were negative (<1 : 25 titer) for antibodies to N. caninum as determined by the indirect fluorescent antibody test (IFAT) (Cole et al., 1995). Both dogs were vaccinated against the common pathogens of dogs (distemper, adenovirus type-2, parainfluenza, parvovirus, and Leptospira) and were treated with fenbendazole at 50 mg/kg for 3 days to remove nematodes. Tapeworm segments of Dipylidium caninum were observed in the feces after the study was initiated but the dogs were not given an anticestocidal agent. Dog 1 was given 100 mg methylprednisolone acetate (MPA) intramuscularly (IM) on days −7, −6, 0, and 1 post-feeding (PF) of infected mouse brains. Each dog was fed 3.8 g of mouse brains that contained tissue cysts of the NC-beef isolate of N. caninum (see below). The brains were placed in a bowl that did not contain food and were consumed within 30 s by each dog. Blood was collected from each dog on Days 0, 3, 7, 10, 14, 21, 28, 35, and 42 days PF, the serum separated, and examined for IgG antibodies in the IFAT. Sera were examined at an initial dilution of 1 : 25 and then endpoint titrated by doubling dilutions. Fecal samples were collected on days −2, −1, 0, 3–21 PF and examined for N. caninum oocysts after flotation in Sheather’s sugar solution (Ernst and Benz, 1981). The dogs were euthanatized by intravenous overdose of sodium pentobarbital on day 42 PF. Portions of tissues from brain, eyes, heart, tongue, lung, liver, spleen, kidney, adrenal gland, mesenteric lymph nodes, skeletal muscles, and intestines were collected, fixed in 10% neutral buffered formalin solution, and processed through graded ethanols, a xylene substitute, and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin. Unstained sections of tissues with lesions were examined for the presence of N. caninum using a mouse monoclonal antibody based immunohistochemical test (Cole et al., 1993). D.S. Lindsay et al. / Veterinary Parasitology 82 (1999) 327–333 329 2.2. Inoculation and examination of mice Twenty Swiss-Webster, 30 g, female mice were immunosuppressed with MPA and each inoculated subcutaneously with 1 × 105 tachyzoites of the NC-beef isolate of N. caninum to obtain infected mouse brain for the dog feeding studies (McGuire et al., 1997). Mice were killed by cervical dislocation 6 months later and their brains removed and used in the present study. Two female ICR mice were each treated with 4 mg MPA IM on Days 7, and 0 prior to each being fed 5 × 104 oocysts by gavage and being injected subcutaneously with 5 × 104 oocysts. These mice received an additional 8 mg MPA IM on Day 7 after feeding/injection of oocysts. The MPA was used because it enhances the susceptibility of mice to neosporo- sis (Lindsay and Dubey, 1989, 1990). Two female BALB/c mice were each fed 1 × 105 oocysts by gavage. The BALB/c mice were used because they are naturally susceptible to encephalitis cause by N. caninum (Lindsay et al., 1995; Long et al., 1998). Eight ␥- INF-KO mice were each fed 1 × 104 oocysts. The ␥-INF-KO mice were used because they have been proven to be susceptible to infection with N. caninum oocysts (McAllister et al., 1998). Surviving mice were bled 6–10 weeks after they were fed oocysts and their sera tested for IgG antibodies to N. caninum in the IFAT at a 1 : 25 dilution. The mice were then killed by cervical dislocation and their brains removed. Two 2 mm3 portions of cerebrum were placed on glass microscope slides, and each compressed with a 22 mm2 glass coverslip, and examined unstained for N. caninum tissue cysts by light microscopy. The remaining portion of brain was fixed in 10% neutral buffered formalin solution and processed for histological examination, as above. A complete necropsy was carried out on all mice that died. Portions of tissues from the heart, tongue, lung, liver, spleen, kidney, adrenal gland, mesenteric lymph nodes, and skele- tal muscles were collected, fixed in 10% neutral buffered formalin solution, and processed for histological examination, as above. Unstained sections of tissues were examined for N. caninum using a mouse monoclonal antibody based immunohistochemical test (Cole et al., 1993). 2.3. Oocyst collection, processing, and sporulation Feces from Dog 1 in which oocysts were detected were collected, mixed in 2% (v/v) sulfuric acid, strained with a tea strainer, placed in 2 l plastic beverage bottles and the suspension aerated with air from an aquarium pump for 48–72 h. Other fresh fecal samples were mixed in 2% (v/v) sulfuric acid and placed in a 4 mm layer in two 150 mm2 vented cell culture flasks and used to examine sporulation after 24 h. One flask was incubated at 37◦C and the other at room temperature (22–25◦C). The daily processed fecal output of Dog 1 on Days 5 to 10 was combined, mixed, and a 300 ml sample taken and concentrated by centrifugation and flotation in Sheather’s sugar solution to estimate the total number of oocysts collected from Dog 1. Oocyst numbers were determined by counting in a hemocytometer and the total numbers of oocyst collected from Dog 1 was then calculated. 330 D.S. Lindsay et al. / Veterinary Parasitology 82 (1999) 327–333 3. Results 3.1. Infections in dogs Dog 1 began excreting N. caninum oocysts 5 days PF and Dog 2 dog began excreting oocysts 6 days PF. Oocysts were detected in the feces of Dog 1 on Days 5 through 10 and on Day 17 PF. Feces from this dog were collected and processed to sporulate and isolate oocysts. Dog 2 had detectable oocysts only on Day 6 and Day 9 PF. Its feces was not used for oocyst collection because of the few oocysts present. Neither dog had diarrhea. Both dogs had normal appetites and were active throughout the period of the study. Dog 1 developed detectable IgG antibodies 35 days PF. The titer was 1 : 200 on this day. Her titer on Day 42 PF was 1 : 1600. Dog 2 did not have detectable IgG antibodies to N. caninum in a 1 : 25 dilution of serum on any examination period. Gross examination of Dog 1 at necropsy revealed small pin-point red foci randomly distributed throughout the lungs and an enlarged, pale red to gray liver.