Confirmation That the Dog Is a Definitive Host for Neospora Caninum

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]

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 neosporosis 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 neosporosis (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 skeletal 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. No gross lesions were observed in Dog 2. In the lungs, livers and intestines microscopic examination of both dogs mild lesions were observed except for those in the lungs of Dog 1 which were considered to be moderate. Lung lesions in both dogs consisted of multifocal pyogranulomatous interstitial pneumonia. Microgranulomas were observed in the parenchyma of the livers of both dogs and in addition hepatocytes in the liver of Dog 1 were vacuolated. In the heart of Dog 1 there were multiple foci of myocarditis characterized by focal inﬁltrates of lymphocytes and macrophages. Lesions in the pancreas were limited to interstitial inﬂammation with mixtures of lymphocytes, plasma cells, and macrophages while lesions in the kidney consisted of mild lymphoplasmacitic nephritis. There were increased numbers of eosinophils in the intestines of both dogs. Neospora caninum stages were not observed in any tissue examined microscopically in either dog. No microscopic lesions were observed in the brains, eyes, or skeletal muscles of Dogs 1 and 2.

3.2. Infections in mice

Neither ICR mouse given MPA and N. caninum oocysts died or developed clinical signs. No tissue cysts were seen in direct smears of their brains. Both mice had antibody titers of 1 : 50 in the IFAT. Lesions consistent with N. caninum infection were observed in histological sections of brains of both mice (Lindsay et al., 1995) and N. caninum tachyzoites were observed in one of the mice using immunohistochemistry. Neither BALB/c mouse fed N. caninum oocysts died and the only clinical sign observed was a slight ruf- ﬂing of the fur coat. One mouse had an antibody titer of 1 : 50 and the other had an antibody titer of 1 : 100 in the IFAT. No tissue cysts were seen in direct smears of their brains. No lesions or N. caninum organisms were observed in brain sections from these mice. One of the ␥-INF-KO mice died 14 days PI. Neospora caninum tachyzoites were observed in the liver, lungs, and spleen by immunohistochemistry (Fig. 1). None of the remaining 7 ␥-INF-KO mice died, seroconverted (<1 : 25 titer) in the IFAT or had detectable parasites in their tissues. No tissue cysts were seen in direct smears of their brains 74 days PI. D.S. Lindsay et al. / Veterinary Parasitology 82 (1999) 327–333 331

Fig. 1. A compact group of immunologically labeled tachyzoites (arrow) and a dispersing group of immunoreactive tachyzoites (arrowhead) next to an area of necrosis (N) present in the liver of a ␥-interferon knockout mouse fed Neospora caninum oocysts. Immunohistochemical method, ×75.

3.3. Oocyst sporulation

More than 95% of the oocysts collected and aerated in plastic beverage bottles for 48–72 h were sporulated when examined microscopically. Some oocysts had sporulated within 24 h at 37◦C or at room temperature. Most oocysts were Isospora-like and contained two sporo- cysts each with four sporozoites, but occasionally, Caryospora-like oocysts with one sporo- cyst and eight sporozoites were observed. The total numbers of oocysts collected from processed feces of Dog 1 on Days 5 through to 10 PF were estimated to be 4.5 × 106. The separation of oocysts from feces was hindered by particulate matter that ﬂoated up with the oocysts and made it difﬁcult to count the oocysts in the hemocytometer.

4. Discussion

Our study conﬁrms that the dog is a deﬁnitive host for N. caninum and extends our knowl- edge about the behavior of this parasite in dogs fed tissue cysts. Our study demonstrated that dogs excrete oocysts following a prepatent period of 5 days. This is 3 days shorter than the previous report (McAllister et al., 1998). The lack of seroconversion in Dog 2 following excretion of oocysts is interesting and has been demonstrated previously with this isolate of N. caninum (McAllister et al., 1998). Dog 2 excreted only a few oocysts on Days 6 and 9 suggesting infection was not as intense as that in Dog 1 which excreted oocysts for 6 consecutive days. The IFAT used measures only IgG antibodies to tachyzoite antigens (Cole et al., 1995). It is possible that no or only minimal extraintestinal infection occurred in this dog and therefore no IgG antibodies to tachyzoites were produced. If this lack of seroconversion by dogs excreting oocysts occurs with other strains of N. caninum 332 D.S. Lindsay et al. / Veterinary Parasitology 82 (1999) 327–333

then the estimates of prevalence of this parasite in dogs based on serological tests will under represent its true prevalence. Neither dog developed obvious clinical signs during the period of oocyst excretion. This was also the case in dogs examined by McAllister et al. (1998). The lesions observed in the livers of both dogs and the heart of Dog 1 are suggestive of neosporosis (Dubey and Lindsay, 1996). The vacuolated hepatocytes in the liver in Dog 1 was probably due to MPA treatment (Jones et al., 1997). The other lesions observed were mild and are considered incidental findings due to the random source nature of the experimental animals. BALB/c and MPA-treated mice were resistant to disease following oral infection with N. caninum oocysts of the NC-beef isolate. Additionally, fatal infection occurred in only 1 of 8 ␥-INF-KO mice further suggesting a lack of pathogenicity of the oocysts of the NC-beef isolate for mice. These observations need to be confirmed using additional isolates of N. caninum oocysts. We determined that N. caninum oocysts sporulate within 24 h under optimal conditions. This is similar to what is observed for many Isospora species and Toxoplasma gondii (Lindsay et al., 1997). McAllister et al. (1998) did not record finding Caryospora-like oocysts of N. caninum. However, our observation of them is perhaps not unexpected as they commonly occur with Isospora species (Lindsay et al., 1997). Dog 1 given MPA excreted more oocysts and excreted oocysts for a longer period than did Dog 2 suggesting this treatment may be a useful technique for research into transmission studies with N. caninum in dogs.

Acknowledgements

The authors thank Susan King, Catherine Walsh, and Kay Carlson for technical assistance.

References

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