- Email: [email protected]
Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum
Veterinary Parasitology 97 (2001) 145–152
Short communication
Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum Nadia Bergeron a , Gilles Fecteau a,∗ , Alain Villeneuve b , Christiane Girard b , Julie Paré c a
b
Faculté de Médecine Vétérinaire, Département de Sciences Cliniques, Université de Montréal, C.P. 5000, St-Hyacinthe, Qué., Canada J2S 7C6 Faculté de Médecine Vétérinaire, Département de Pathologie et Microbiologie, Université de Montréal, C.P. 5000, St-Hyacinthe, Qué., Canada J2S 7C6 c Biovet, 2900 rue Vanier, St-Hyacinthe, Qué., Canada J2S 6M2 Received 31 August 2000; received in revised form 29 January 2001; accepted 22 February 2001
Abstract Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2–4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum. © 2001 Published by Elsevier Science B.V. Keywords: Neospora caninum; Dogs; Cattle; Definitive host
1. Introduction Neospora caninum principally causes abortion in dairy cattle (Barr et al., 1990, 1991a; Anderson et al., 1991, 1995; Dubey, 1999a). The entire life cycle of N. caninum is still unclear. Until recently, the only stages identified were tachyzoites and cysts containing bradyzoites. McAllister et al. (1998) showed the presence of oocysts of N. caninum in the feces of dogs which had ingested cysts of N. caninum experimentally produced in mice. Seroepidemiological studies support the role of dogs in the cycle of N. caninum (Paré et al., 1998; Sawada et al., 1998; Wouda et al., 1999). The presence and the number of ∗ Corresponding author. Tel.: +1-450-773-8521/ext. 8337; fax: +1-450-778-8102. E-mail address: [email protected] (G. Fecteau).
0304-4017/01/$ – see front matter © 2001 Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 3 9 6 - X
146
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
dogs are correlated to high seroprevalence of N. caninum in dairy cattle (Paré et al., 1998; Wouda et al., 1999). The seroprevalence was found to be higher in dogs living on dairies as compared to dogs living in urban area (Sawada et al., 1998; Wouda et al., 1999). A possible source of N. caninum for the final host is aborted bovine fetuses or dead infected calves. The objective of this study was to verify whether dogs fed on infected bovine fetuses containing N. caninum excrete oocysts and seroconvert against this agent. 2. Materials and methods Several Québec large animal veterinary clinics were solicited to obtain bovine fetuses from cows serologically positive to N. caninum or from herds known to have N. caninum infected animals. When the serological status of the cow was unknown, a blood sample from the cow was submitted with the aborted fetus to determine its serological status by enzyme-linked immunosorbent assay (ELISA) testing (Biovet, St-Hyacinthe, Qué., Canada). Three or four pieces of fetal brain were examined histopathologically to identify the presence of N. caninum or changes suggestive of an infection by this agent. Selected paraffin blocks were examined by immunoperoxidase (IP) (Vector Laboratories, Burlingame, CA) using standard technique (Lindsay and Dubey, 1989) (Fig. 1). Only brain was examined because asexual stages of N. caninum are more common in neural tissues of aborted bovine fetuses than in other tissues (Dubey and Lindsay, 1996). Twelve 2–4-month-old healthy dogs were randomly allotted in three groups, each of them comprising three inoculated animals and one control (Table 1). Dogs were housed in individual metal cages and fed 400 g of commercial growth feed daily. On day 0, dogs were fed on infected fetuses. Sample collection and observations were performed for a period of 6 weeks. Physical examination included: rectal temperature measurements, heart and respiratory rates, weight and a subjective evaluation of alertness.
Fig. 1. N. caninum cyst shown in the brain of an aborted bovine fetus by immunohistochemistry. Bar = 20 m.
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
147
Table 1 Demographic data, ELISA ratio and coprology result of the dogs of the three experiments on arrival Dog identification
Age (week)
Weight (kg)
Sex
Breed
ELISA ratiob
Coprology
Acclimation (day)
Control A 1 2 3
11 9 11 9
11.0 11.6 8.8 9.8
M M M M
MBa MB MB MB
0.00 0.03 0.02 0.02
Isospora Negative Toxocara Toxocara
14 14 14 14
Control B 4 5 6
8 9 7 8
7.0 6.0 6.2 4.8
M M M M
MB MB MB MB
0.02 0.08 0.01 0.03
Isospora, Toxocara Toxocara Toxocara Negative
21 21 25 23
Control C 7 8 9
8 8 8 8
3.4 4.0 2.6 2.2
F M F F
MB MB Pitbull Pitbull
0.00 0.00 0.00 0.00
Isospora, Toxocara Isospora, Toxocara Isospora, Toxocara Isospora, Toxocara
8 8 22 44
a b
Mixed-breed. Positive if ratio ≥ 0.30.
ELISA for N. caninum was done 3 and 6 weeks post-inoculation. Results were considered positive if the ratio was ≥0.30 with a sensitivity and a specificity of ≥99% (Biovet, St-Hyacinthe, Qué., Canada). A blood sample with EDTA was taken twice a week for a complete blood count (CBC). Coprological analyses were performed using the Wisconsin method (Cox and Todd, 1962) every 2 days for the first 2 weeks, everyday for the third week and every 2 days for the remaining 3 weeks, based on experimental observations (McAllister et al., 1998). Feces of each animal were collected once daily, mixed, and 2 g were sampled for analysis. Fecal samples were stored at 4◦ C until analyzed. At the end of the study, the dogs were euthanized by injection (0.3 ml/kg of body weight of 340 mg/ml of Euthansol® , Schering-Plough Animal Health, Division of Schering Canada Inc., Qué., Canada). A necropsy was performed on each animal. Pieces of neural system, muscles, thymus, hearth, lung, liver, spleen, pancreas, kidney, intestine and lymph nodes were sampled, fixed in 10% buffered formalin, embedded in paraffin, cut at 6 m and stained with hematoxylin-phoxin-saffron (HPS) for histopathological examination. 3. Results 3.1. First experiment For the first group (control A, dogs 1, 2 and 3), histopathological examination and IP were performed on the brain of all fetuses from seropositive dams. The three dogs were fed the same day an equal portion of five brains from infected fetuses (4–9 months of gestation) stored at −70◦ C for 4–28 days and a placenta from a seropositive dam (Table 2). The physical examination and the CBC of the dogs in the first group did not revealed any change. None of the dogs developed antibodies against N. caninum or shed detectable
148
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
Table 2 ELISA ratio of the aborted N. caninum seropositive cows and results of the examination of the fetal brains for the three experiments Dog identification Number of days that dogs kept in the study
Fetusa
ELISA ratiob
Histopathological examinationc
Immunoperoxidase (IP)c
1, 2 and 3
43
A B C D + pd E
1.26 1.44 2.16 1.50 1.22
No lesion No lesion Granulomatous foci No lesion No lesion
Negative Cysts Tachyzoites and cysts Negative Negative
4
43
F G
1.68 1.74
No lesion Tachyzoites Granulomatous foci Tachyzoites
5
43
H I
1.32 0.69
No lesion No lesion
6
43
J K
1.38 0.73
Granulomatous foci Tachyzoites No lesion Negative
7
59
L M N
1.41 1.01 0.84
Granulomatous foci Tachyzoites and cysts Granulomatous foci Tachyzoites and cysts Granulomatous foci Tachyzoites
8e
52
O P Q
1.57 0.71 1.10
Granulomatous foci Negative No lesion Negative No lesion Negative
9
65
R S T
0.63 1.98 1.61
No lesion Negative Granulomatous foci Tachyzoites and cysts Granulomatous foci Tachyzoites and cysts
Tachyzoites Negative
a
Fetuses used to inoculate the corresponding dog(s). ELISA ratio of aborted cows. Ratio ≥0.60 is considered positive. c Results for the brain. d Placenta. e Dog 8 died. b
levels of N. caninum oocysts’. The dogs had, however, oocysts of Isospora (1 to more than 100 oocysts per field) and Cryptosporidium, eggs of Toxocara and Toxascaris and cysts of Giardia. Macroscopic changes were limited to the presence of ascarids in the small intestine of the control A and in dogs 1 and 2. Microscopic changes were limited to the presence of parasites in the lumen of the intestine. 3.2. Second experiment For the second group (control B, dogs 4, 5 and 6), histopathological examination and IP were performed on the brain of each fetus submitted. The fetuses were stored at 4◦ C, and fed to dogs as soon as the results became available (2–4 weeks). Dogs were infected sequentially. Each dog received two fetuses considered infected with N. caninum. Fetuses were considered infected if they had lesions compatible with an infection by N. caninum on histopathological or positive IP results or if the dam was seropositive to N. caninum.
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
149
The physical exam and CBC of the dogs in the second group revealed no significant anomaly. None of the dogs developed antibodies against N. caninum or shed detectable levels of N. caninum oocysts’. The dogs, however, had oocysts of Isospora (1–100 oocysts per field), eggs of Toxocara and Toxascaris and cysts of Giardia. Macroscopic changes were limited to the presence of ascarids in the small intestine for dog 5. Small intrasinusoidal granulomas were observed in the liver of dog 6 and were possibly caused by migration of ascarid. 3.3. Third experiment Fetuses were given to the dogs of the last group (control C, dogs 7, 8 and 9) as soon as they were submitted, before the results of any test were available. A histopathological examination was performed on the brains of all fetuses. An IP test was also performed on the brains of all fetuses from seropositive dams. The inoculation was performed sequentially. One of the two following criteria had to be met before a dog was considered to have received sufficient infectious material: (1) ingestion of four fetuses from seropositive dams, having brain lesions compatible with an infection with N. caninum on histopathological examination and/or a positive IP result to N. caninum or, (2) ingestion of at least one fetal brain containing cysts shown by an IP test. When a dog had met one of the two criteria, the 6 weeks of observation began. However, a maximum infection period of 3 weeks was allowed for each dog, regardless of the number of fetuses ingested. Finally, nine fetuses were determined positive for this experiment. To increase its susceptibility to infection, dog 9 received 4 mg/kg per os once a day of prednisone (Apo® -Prednisone, Apotex Inc., Ont., Canada) for the duration of the infection period (Lindsay et al., 1999). The administration of prednisone began the day prior to the ingestion of the first fetus and ended 4 days post-ingestion of the last fetus. Over the course of the experiment, control C developed gastrointestinal clinical signs compatible with parvoviral enteritis. Dog 7 developed episodes of hyperthermia (39.3 to 39.6◦ C) associated with excitement or with an elevated ambient temperature. Dog 8 developed several periods of hyperthermia (39.3 to 39.7◦ C) followed by fatal gastroenteritis compatible with parvoviral infection. Dog 9 had a decreased appetite for 1 week and several episodes of hyperthermia (39.5 to 40.1◦ C) without other clinical signs. None of the dogs in the third group developed antibodies against N. caninum. The only CBC change encountered in control C was an eosinophilia probably associated with intestinal parasites. Dog 7 developed a leukocytosis, possibly caused by stress or parasitism. Dog 8 did not have significant CBC changes during the experiment. Dog 9 had lymphopenia and a significant neutropenia that may be associated with a subclinical parvoviral infection. No oocyst of N. caninum was detected. The dogs of this group had oocysts of Isospora (1 to more than 100 oocysts per field), eggs of Toxocara and Trichuris and cysts of Giardia. Microscopic changes (granulomatous foci in the liver for control C, dogs 7 and 9) could be a result of the migration of ascarid. Indeed, control C, dogs 7 and 9 shed Toxocara eggs throughout most of the experiment. The microscopic lesions of dog 8 were compatible with acute parvoviral enteritis.
150
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
4. Discussion The failure to produce infection in dogs in these experiments may be caused by the storage of fetuses resulting in a decrease of their infectivity. The time elapsed between abortion and arrival of the fetus at the Faculté de Médecine Vétérinaire varied from 1 to 5 days. In addition, fetuses were stored at −70◦ C for the first group and 4◦ C for the second group. The delay at various temperatures may have influenced the infectious potential of the fetuses. Bradyzoites in homogenized brain of mouse are killed by freezing at −20◦ C for 7 days (Lindsay et al., 1992). However, cysts from a calf’s brain survived at −52◦ C for about 4 months (Bryan et al., 1994). Bradyzoites in cysts survived at least 14 days at 4◦ C in a homogenized brain and they also survived in the intact brain of a mouse carcass at 4◦ C for 7 days (Lindsay et al., 1992). For group 3, the fetuses were fed to the dogs as soon as they were received to reduce the delay between abortion and ingestion by dogs. Despite this change in the protocol, no dog was infected. Another explanation for the unsuccessful infection may be a low number of parasites in the fetuses. The precise number of parasites (tachyzoites and cysts) present in infected fetuses was not determined in this study. It appears to be difficult to isolate parasites from fetal brains, suggesting that parasites present are few in number or non viable (Dubey and Lindsay, 1996; Conrad et al., 1993). In the experiment of McAllister et al. (1998), the number of cysts given to each dog varied from 125 to 300. However, the concentration of oocysts in their feces was low and seemed to be roughly in proportion to the number of cysts ingested by the dogs (McAllister et al., 1998). In our experiment, cysts were only identified in 6 fetuses. If the inoculum contained only tachyzoites, they may have been be destroyed by gastric digestion (McAllister, 1999). Also, some fetuses may not have been infected in retrospect. Four fetuses (I, K, P and R) originated from dams barely over the seropositivity threshold, and did not bear indication of infection by histopathological or IP examinations. However, low sensitivity of the IP (Dubey, 1999b) may have decreased the number of apparently infected fetuses. Considering low sensitivity of IP, all material fed may potentially have been infected. More cysts seem to be present in the spinal cord than in the brain (Barr et al., 1991b). In fetuses congenitally infected, N. caninum is confined to the brain and the spinal cord (Dubey and Lindsay, 1996). In this experiment, the dogs received all the fetal brains but only the cranial third or less of the spinal cord, which may have reduced the number of parasites given. The small number of cysts ingested could have prevented the infection or the number of oocysts in the feces may have been so small that they could not be identified. McAllister (1999) reports that the number of oocysts observed in feces may be affected by the number of cysts ingested and the type and quantity of food given to inoculate dogs. According to the studies of McAllister et al. (1998) and Lindsay et al. (1999), dogs may excrete oocysts without detectable seroconversion. However, the necropsy may have confirmed the presence of infection, since according to the study of Lindsay et al. (1999), two out of two dogs inoculated had lesions compatible with an infection from N. caninum. If dogs become infected by eating contaminated fetuses on farm, it is likely that they ingest relatively fresh infectious material. However, it is unlikely that they are exposed to multiple fetuses in a short period of time performed in this experiment. Perhaps, natural
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
151
exposure occurs at a specific time of their life which may correspond to a reduced capacity of their immunological system. It is also possible that the dog is not the definitive host of N. caninum, but an accidental one. The lack of infection in dogs through infected bovine fetuses and the reported low level of oocyst shedding in dogs experimentally infected with mice (McAllister et al., 1998), suggest the true definitive host may be a canid other than the dog. The hypothesis that a wild canid be the true definitive host of N. caninum, must be investigated. The successful infection by McAllister et al. (1998) and Lindsay et al. (1999) using mice raises the hypothesis that a second intermediate host may be necessary for completion of the cycle. This second intermediate host would become infected by ingesting bovine fetuses and the dog in turn would ingest this intermediate host to become infected. Farm dogs are likely to eat small mammals and birds which possibly contain N. caninum tissue cysts (Wouda et al., 1999). This hypothetical cycle would explain why in the studies of McAllister et al. (1998) and Lindsay et al. (1999) dogs excreted oocysts and that following an inoculation with bovine fetuses, there was no apparent infection. In conclusion, the fact that it was not possible to infect dogs with bovine fetuses considered infected by N. caninum suggests that the cycle of this parasite is not completely understood. Indeed, the role of the dog as definitive host has not been shown except when infected with experimentally infected mice. Other studies will be required to clarify the role of dogs and wild canids in the epidemiology of this disease.
Acknowledgements We thank veterinarians who participated to this study by sending fetuses. This research was supported by the Fonds du Centenaire from the Faculté de Médecine Vétérinaire of the Université de Montréal. References Anderson, M.L., Blanchard, P.C., Barr, B.C., Dubey, J.P., Hoffman, R.L., Conrad, P.A., 1991. Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. J. Am. Vet. Med. Assoc. 198, 241–244. Anderson, M.L., Palmer, C.W., Thurmond, M.C., Picanso, J.P., Blanchard, P.C., Breitmeyer, R.E., Layton, A.W., McAllister, M., Daft, B., Kinde, H., Read, D.H., Dubey, J.P., Conrad, P.A., Barr, B.C., 1995. Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. J. Am. Vet. Med. Assoc. 207, 1206–1210. Barr, B.C., Anderson, M.L., Blanchard, P.C., Daft, B.M., Kinde, H., Conrad, P.A., 1990. Bovine fetal encephalitis and myocarditis associated with protozoal infections. Vet. Pathol. 27, 354–361. Barr, B.C., Anderson, M.L., Dubey, J.P., Conrad, P.A., 1991a. Neospora-like protozoal infections associated with bovine abortions. Vet. Pathol. 28, 110–116. Barr, B.C., Conrad, P.A., Dubey, J.P., Anderson, M.L., 1991b. Neospora-like encephalomyelitis in a calf: pathology, ultrastructure, and immunoreactivity. J. Vet. Diagn. Invest. 3, 39–46. Bryan, L.A., Gajadhar, A.A., Dubey, J.P., Haines, D.M., 1994. Bovine neonatal encephalomyelitis associated with a Neospora sp. protozoan. Can. Vet. J. 35, 111–113. Conrad, P.A., Barr, B.C., Sverlow, K.W., Anderson, M., Daft, B., Kinde, H., Dubey, J.P., Munson, L., Ardans, A., 1993. In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses. Parasitology 106, 239–249.
152
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
Cox, D.D., Todd, A.C., 1962. Survey of gastrointestinal parasitism in Wisconsin dairy cattle. J. Am. Vet. Med. Assoc. 141, 706–709. Dubey, J.P., 1999a. Neosporosis in cattle: biology and economic impact. J. Am. Vet. Med. Assoc. 214, 1160–1163. Dubey, J.P., 1999b. Recent advances in Neospora and neosporosis. Vet. Parasitol. 84, 349–367. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Lindsay, D.S., Dubey, J.P., 1989. Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am. J. Vet. Res. 50, 1981–1983. Lindsay, D.S., Blagburn, B.L., Dubey, J.P., 1992. Factors affecting the survival of Neospora caninum bradyzoites in murine tissues. J. Parasitol. 78, 70–72. Lindsay, D.S., Dubey, J.P., Duncan, R.B., 1999. Confirmation that the dog is a definitive host for Neospora caninum. Vet. Parasitol. 82, 327–333. McAllister, M.M., 1999. Uncovering the biology and epidemiology of Neospora caninum. Parasitol. Today 15, 216–217. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. Paré, J., Fecteau, G., Fortin, M., Marsolais, G., 1998. Seroepidemiologic study of Neospora caninum in dairy herds. J. Am. Vet. Med. Assoc. 213, 1595–1598. Sawada, M., Park, C., Kondo, H., Morita, T., Shimada, A., Yamane, I., Umenura, T., 1998. Serological survey of antibody to Neospora caninum in Japanese dogs. J. Vet. Med. Sci. 60, 853–854. Wouda, W., Dijkstra, Th., Kramer, A.M.H., van Maanen, C., Brinkhof, J.M.A., 1999. Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle. Int. J. Parasitol. 29, 1677–1682.
Short communication
Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum Nadia Bergeron a , Gilles Fecteau a,∗ , Alain Villeneuve b , Christiane Girard b , Julie Paré c a
b
Faculté de Médecine Vétérinaire, Département de Sciences Cliniques, Université de Montréal, C.P. 5000, St-Hyacinthe, Qué., Canada J2S 7C6 Faculté de Médecine Vétérinaire, Département de Pathologie et Microbiologie, Université de Montréal, C.P. 5000, St-Hyacinthe, Qué., Canada J2S 7C6 c Biovet, 2900 rue Vanier, St-Hyacinthe, Qué., Canada J2S 6M2 Received 31 August 2000; received in revised form 29 January 2001; accepted 22 February 2001
Abstract Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2–4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum. © 2001 Published by Elsevier Science B.V. Keywords: Neospora caninum; Dogs; Cattle; Definitive host
1. Introduction Neospora caninum principally causes abortion in dairy cattle (Barr et al., 1990, 1991a; Anderson et al., 1991, 1995; Dubey, 1999a). The entire life cycle of N. caninum is still unclear. Until recently, the only stages identified were tachyzoites and cysts containing bradyzoites. McAllister et al. (1998) showed the presence of oocysts of N. caninum in the feces of dogs which had ingested cysts of N. caninum experimentally produced in mice. Seroepidemiological studies support the role of dogs in the cycle of N. caninum (Paré et al., 1998; Sawada et al., 1998; Wouda et al., 1999). The presence and the number of ∗ Corresponding author. Tel.: +1-450-773-8521/ext. 8337; fax: +1-450-778-8102. E-mail address: [email protected] (G. Fecteau).
0304-4017/01/$ – see front matter © 2001 Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 3 9 6 - X
146
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
dogs are correlated to high seroprevalence of N. caninum in dairy cattle (Paré et al., 1998; Wouda et al., 1999). The seroprevalence was found to be higher in dogs living on dairies as compared to dogs living in urban area (Sawada et al., 1998; Wouda et al., 1999). A possible source of N. caninum for the final host is aborted bovine fetuses or dead infected calves. The objective of this study was to verify whether dogs fed on infected bovine fetuses containing N. caninum excrete oocysts and seroconvert against this agent. 2. Materials and methods Several Québec large animal veterinary clinics were solicited to obtain bovine fetuses from cows serologically positive to N. caninum or from herds known to have N. caninum infected animals. When the serological status of the cow was unknown, a blood sample from the cow was submitted with the aborted fetus to determine its serological status by enzyme-linked immunosorbent assay (ELISA) testing (Biovet, St-Hyacinthe, Qué., Canada). Three or four pieces of fetal brain were examined histopathologically to identify the presence of N. caninum or changes suggestive of an infection by this agent. Selected paraffin blocks were examined by immunoperoxidase (IP) (Vector Laboratories, Burlingame, CA) using standard technique (Lindsay and Dubey, 1989) (Fig. 1). Only brain was examined because asexual stages of N. caninum are more common in neural tissues of aborted bovine fetuses than in other tissues (Dubey and Lindsay, 1996). Twelve 2–4-month-old healthy dogs were randomly allotted in three groups, each of them comprising three inoculated animals and one control (Table 1). Dogs were housed in individual metal cages and fed 400 g of commercial growth feed daily. On day 0, dogs were fed on infected fetuses. Sample collection and observations were performed for a period of 6 weeks. Physical examination included: rectal temperature measurements, heart and respiratory rates, weight and a subjective evaluation of alertness.
Fig. 1. N. caninum cyst shown in the brain of an aborted bovine fetus by immunohistochemistry. Bar = 20 m.
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
147
Table 1 Demographic data, ELISA ratio and coprology result of the dogs of the three experiments on arrival Dog identification
Age (week)
Weight (kg)
Sex
Breed
ELISA ratiob
Coprology
Acclimation (day)
Control A 1 2 3
11 9 11 9
11.0 11.6 8.8 9.8
M M M M
MBa MB MB MB
0.00 0.03 0.02 0.02
Isospora Negative Toxocara Toxocara
14 14 14 14
Control B 4 5 6
8 9 7 8
7.0 6.0 6.2 4.8
M M M M
MB MB MB MB
0.02 0.08 0.01 0.03
Isospora, Toxocara Toxocara Toxocara Negative
21 21 25 23
Control C 7 8 9
8 8 8 8
3.4 4.0 2.6 2.2
F M F F
MB MB Pitbull Pitbull
0.00 0.00 0.00 0.00
Isospora, Toxocara Isospora, Toxocara Isospora, Toxocara Isospora, Toxocara
8 8 22 44
a b
Mixed-breed. Positive if ratio ≥ 0.30.
ELISA for N. caninum was done 3 and 6 weeks post-inoculation. Results were considered positive if the ratio was ≥0.30 with a sensitivity and a specificity of ≥99% (Biovet, St-Hyacinthe, Qué., Canada). A blood sample with EDTA was taken twice a week for a complete blood count (CBC). Coprological analyses were performed using the Wisconsin method (Cox and Todd, 1962) every 2 days for the first 2 weeks, everyday for the third week and every 2 days for the remaining 3 weeks, based on experimental observations (McAllister et al., 1998). Feces of each animal were collected once daily, mixed, and 2 g were sampled for analysis. Fecal samples were stored at 4◦ C until analyzed. At the end of the study, the dogs were euthanized by injection (0.3 ml/kg of body weight of 340 mg/ml of Euthansol® , Schering-Plough Animal Health, Division of Schering Canada Inc., Qué., Canada). A necropsy was performed on each animal. Pieces of neural system, muscles, thymus, hearth, lung, liver, spleen, pancreas, kidney, intestine and lymph nodes were sampled, fixed in 10% buffered formalin, embedded in paraffin, cut at 6 m and stained with hematoxylin-phoxin-saffron (HPS) for histopathological examination. 3. Results 3.1. First experiment For the first group (control A, dogs 1, 2 and 3), histopathological examination and IP were performed on the brain of all fetuses from seropositive dams. The three dogs were fed the same day an equal portion of five brains from infected fetuses (4–9 months of gestation) stored at −70◦ C for 4–28 days and a placenta from a seropositive dam (Table 2). The physical examination and the CBC of the dogs in the first group did not revealed any change. None of the dogs developed antibodies against N. caninum or shed detectable
148
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
Table 2 ELISA ratio of the aborted N. caninum seropositive cows and results of the examination of the fetal brains for the three experiments Dog identification Number of days that dogs kept in the study
Fetusa
ELISA ratiob
Histopathological examinationc
Immunoperoxidase (IP)c
1, 2 and 3
43
A B C D + pd E
1.26 1.44 2.16 1.50 1.22
No lesion No lesion Granulomatous foci No lesion No lesion
Negative Cysts Tachyzoites and cysts Negative Negative
4
43
F G
1.68 1.74
No lesion Tachyzoites Granulomatous foci Tachyzoites
5
43
H I
1.32 0.69
No lesion No lesion
6
43
J K
1.38 0.73
Granulomatous foci Tachyzoites No lesion Negative
7
59
L M N
1.41 1.01 0.84
Granulomatous foci Tachyzoites and cysts Granulomatous foci Tachyzoites and cysts Granulomatous foci Tachyzoites
8e
52
O P Q
1.57 0.71 1.10
Granulomatous foci Negative No lesion Negative No lesion Negative
9
65
R S T
0.63 1.98 1.61
No lesion Negative Granulomatous foci Tachyzoites and cysts Granulomatous foci Tachyzoites and cysts
Tachyzoites Negative
a
Fetuses used to inoculate the corresponding dog(s). ELISA ratio of aborted cows. Ratio ≥0.60 is considered positive. c Results for the brain. d Placenta. e Dog 8 died. b
levels of N. caninum oocysts’. The dogs had, however, oocysts of Isospora (1 to more than 100 oocysts per field) and Cryptosporidium, eggs of Toxocara and Toxascaris and cysts of Giardia. Macroscopic changes were limited to the presence of ascarids in the small intestine of the control A and in dogs 1 and 2. Microscopic changes were limited to the presence of parasites in the lumen of the intestine. 3.2. Second experiment For the second group (control B, dogs 4, 5 and 6), histopathological examination and IP were performed on the brain of each fetus submitted. The fetuses were stored at 4◦ C, and fed to dogs as soon as the results became available (2–4 weeks). Dogs were infected sequentially. Each dog received two fetuses considered infected with N. caninum. Fetuses were considered infected if they had lesions compatible with an infection by N. caninum on histopathological or positive IP results or if the dam was seropositive to N. caninum.
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
149
The physical exam and CBC of the dogs in the second group revealed no significant anomaly. None of the dogs developed antibodies against N. caninum or shed detectable levels of N. caninum oocysts’. The dogs, however, had oocysts of Isospora (1–100 oocysts per field), eggs of Toxocara and Toxascaris and cysts of Giardia. Macroscopic changes were limited to the presence of ascarids in the small intestine for dog 5. Small intrasinusoidal granulomas were observed in the liver of dog 6 and were possibly caused by migration of ascarid. 3.3. Third experiment Fetuses were given to the dogs of the last group (control C, dogs 7, 8 and 9) as soon as they were submitted, before the results of any test were available. A histopathological examination was performed on the brains of all fetuses. An IP test was also performed on the brains of all fetuses from seropositive dams. The inoculation was performed sequentially. One of the two following criteria had to be met before a dog was considered to have received sufficient infectious material: (1) ingestion of four fetuses from seropositive dams, having brain lesions compatible with an infection with N. caninum on histopathological examination and/or a positive IP result to N. caninum or, (2) ingestion of at least one fetal brain containing cysts shown by an IP test. When a dog had met one of the two criteria, the 6 weeks of observation began. However, a maximum infection period of 3 weeks was allowed for each dog, regardless of the number of fetuses ingested. Finally, nine fetuses were determined positive for this experiment. To increase its susceptibility to infection, dog 9 received 4 mg/kg per os once a day of prednisone (Apo® -Prednisone, Apotex Inc., Ont., Canada) for the duration of the infection period (Lindsay et al., 1999). The administration of prednisone began the day prior to the ingestion of the first fetus and ended 4 days post-ingestion of the last fetus. Over the course of the experiment, control C developed gastrointestinal clinical signs compatible with parvoviral enteritis. Dog 7 developed episodes of hyperthermia (39.3 to 39.6◦ C) associated with excitement or with an elevated ambient temperature. Dog 8 developed several periods of hyperthermia (39.3 to 39.7◦ C) followed by fatal gastroenteritis compatible with parvoviral infection. Dog 9 had a decreased appetite for 1 week and several episodes of hyperthermia (39.5 to 40.1◦ C) without other clinical signs. None of the dogs in the third group developed antibodies against N. caninum. The only CBC change encountered in control C was an eosinophilia probably associated with intestinal parasites. Dog 7 developed a leukocytosis, possibly caused by stress or parasitism. Dog 8 did not have significant CBC changes during the experiment. Dog 9 had lymphopenia and a significant neutropenia that may be associated with a subclinical parvoviral infection. No oocyst of N. caninum was detected. The dogs of this group had oocysts of Isospora (1 to more than 100 oocysts per field), eggs of Toxocara and Trichuris and cysts of Giardia. Microscopic changes (granulomatous foci in the liver for control C, dogs 7 and 9) could be a result of the migration of ascarid. Indeed, control C, dogs 7 and 9 shed Toxocara eggs throughout most of the experiment. The microscopic lesions of dog 8 were compatible with acute parvoviral enteritis.
150
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
4. Discussion The failure to produce infection in dogs in these experiments may be caused by the storage of fetuses resulting in a decrease of their infectivity. The time elapsed between abortion and arrival of the fetus at the Faculté de Médecine Vétérinaire varied from 1 to 5 days. In addition, fetuses were stored at −70◦ C for the first group and 4◦ C for the second group. The delay at various temperatures may have influenced the infectious potential of the fetuses. Bradyzoites in homogenized brain of mouse are killed by freezing at −20◦ C for 7 days (Lindsay et al., 1992). However, cysts from a calf’s brain survived at −52◦ C for about 4 months (Bryan et al., 1994). Bradyzoites in cysts survived at least 14 days at 4◦ C in a homogenized brain and they also survived in the intact brain of a mouse carcass at 4◦ C for 7 days (Lindsay et al., 1992). For group 3, the fetuses were fed to the dogs as soon as they were received to reduce the delay between abortion and ingestion by dogs. Despite this change in the protocol, no dog was infected. Another explanation for the unsuccessful infection may be a low number of parasites in the fetuses. The precise number of parasites (tachyzoites and cysts) present in infected fetuses was not determined in this study. It appears to be difficult to isolate parasites from fetal brains, suggesting that parasites present are few in number or non viable (Dubey and Lindsay, 1996; Conrad et al., 1993). In the experiment of McAllister et al. (1998), the number of cysts given to each dog varied from 125 to 300. However, the concentration of oocysts in their feces was low and seemed to be roughly in proportion to the number of cysts ingested by the dogs (McAllister et al., 1998). In our experiment, cysts were only identified in 6 fetuses. If the inoculum contained only tachyzoites, they may have been be destroyed by gastric digestion (McAllister, 1999). Also, some fetuses may not have been infected in retrospect. Four fetuses (I, K, P and R) originated from dams barely over the seropositivity threshold, and did not bear indication of infection by histopathological or IP examinations. However, low sensitivity of the IP (Dubey, 1999b) may have decreased the number of apparently infected fetuses. Considering low sensitivity of IP, all material fed may potentially have been infected. More cysts seem to be present in the spinal cord than in the brain (Barr et al., 1991b). In fetuses congenitally infected, N. caninum is confined to the brain and the spinal cord (Dubey and Lindsay, 1996). In this experiment, the dogs received all the fetal brains but only the cranial third or less of the spinal cord, which may have reduced the number of parasites given. The small number of cysts ingested could have prevented the infection or the number of oocysts in the feces may have been so small that they could not be identified. McAllister (1999) reports that the number of oocysts observed in feces may be affected by the number of cysts ingested and the type and quantity of food given to inoculate dogs. According to the studies of McAllister et al. (1998) and Lindsay et al. (1999), dogs may excrete oocysts without detectable seroconversion. However, the necropsy may have confirmed the presence of infection, since according to the study of Lindsay et al. (1999), two out of two dogs inoculated had lesions compatible with an infection from N. caninum. If dogs become infected by eating contaminated fetuses on farm, it is likely that they ingest relatively fresh infectious material. However, it is unlikely that they are exposed to multiple fetuses in a short period of time performed in this experiment. Perhaps, natural
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
151
exposure occurs at a specific time of their life which may correspond to a reduced capacity of their immunological system. It is also possible that the dog is not the definitive host of N. caninum, but an accidental one. The lack of infection in dogs through infected bovine fetuses and the reported low level of oocyst shedding in dogs experimentally infected with mice (McAllister et al., 1998), suggest the true definitive host may be a canid other than the dog. The hypothesis that a wild canid be the true definitive host of N. caninum, must be investigated. The successful infection by McAllister et al. (1998) and Lindsay et al. (1999) using mice raises the hypothesis that a second intermediate host may be necessary for completion of the cycle. This second intermediate host would become infected by ingesting bovine fetuses and the dog in turn would ingest this intermediate host to become infected. Farm dogs are likely to eat small mammals and birds which possibly contain N. caninum tissue cysts (Wouda et al., 1999). This hypothetical cycle would explain why in the studies of McAllister et al. (1998) and Lindsay et al. (1999) dogs excreted oocysts and that following an inoculation with bovine fetuses, there was no apparent infection. In conclusion, the fact that it was not possible to infect dogs with bovine fetuses considered infected by N. caninum suggests that the cycle of this parasite is not completely understood. Indeed, the role of the dog as definitive host has not been shown except when infected with experimentally infected mice. Other studies will be required to clarify the role of dogs and wild canids in the epidemiology of this disease.
Acknowledgements We thank veterinarians who participated to this study by sending fetuses. This research was supported by the Fonds du Centenaire from the Faculté de Médecine Vétérinaire of the Université de Montréal. References Anderson, M.L., Blanchard, P.C., Barr, B.C., Dubey, J.P., Hoffman, R.L., Conrad, P.A., 1991. Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. J. Am. Vet. Med. Assoc. 198, 241–244. Anderson, M.L., Palmer, C.W., Thurmond, M.C., Picanso, J.P., Blanchard, P.C., Breitmeyer, R.E., Layton, A.W., McAllister, M., Daft, B., Kinde, H., Read, D.H., Dubey, J.P., Conrad, P.A., Barr, B.C., 1995. Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. J. Am. Vet. Med. Assoc. 207, 1206–1210. Barr, B.C., Anderson, M.L., Blanchard, P.C., Daft, B.M., Kinde, H., Conrad, P.A., 1990. Bovine fetal encephalitis and myocarditis associated with protozoal infections. Vet. Pathol. 27, 354–361. Barr, B.C., Anderson, M.L., Dubey, J.P., Conrad, P.A., 1991a. Neospora-like protozoal infections associated with bovine abortions. Vet. Pathol. 28, 110–116. Barr, B.C., Conrad, P.A., Dubey, J.P., Anderson, M.L., 1991b. Neospora-like encephalomyelitis in a calf: pathology, ultrastructure, and immunoreactivity. J. Vet. Diagn. Invest. 3, 39–46. Bryan, L.A., Gajadhar, A.A., Dubey, J.P., Haines, D.M., 1994. Bovine neonatal encephalomyelitis associated with a Neospora sp. protozoan. Can. Vet. J. 35, 111–113. Conrad, P.A., Barr, B.C., Sverlow, K.W., Anderson, M., Daft, B., Kinde, H., Dubey, J.P., Munson, L., Ardans, A., 1993. In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses. Parasitology 106, 239–249.
152
N. Bergeron et al. / Veterinary Parasitology 97 (2001) 145–152
Cox, D.D., Todd, A.C., 1962. Survey of gastrointestinal parasitism in Wisconsin dairy cattle. J. Am. Vet. Med. Assoc. 141, 706–709. Dubey, J.P., 1999a. Neosporosis in cattle: biology and economic impact. J. Am. Vet. Med. Assoc. 214, 1160–1163. Dubey, J.P., 1999b. Recent advances in Neospora and neosporosis. Vet. Parasitol. 84, 349–367. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Lindsay, D.S., Dubey, J.P., 1989. Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am. J. Vet. Res. 50, 1981–1983. Lindsay, D.S., Blagburn, B.L., Dubey, J.P., 1992. Factors affecting the survival of Neospora caninum bradyzoites in murine tissues. J. Parasitol. 78, 70–72. Lindsay, D.S., Dubey, J.P., Duncan, R.B., 1999. Confirmation that the dog is a definitive host for Neospora caninum. Vet. Parasitol. 82, 327–333. McAllister, M.M., 1999. Uncovering the biology and epidemiology of Neospora caninum. Parasitol. Today 15, 216–217. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. Paré, J., Fecteau, G., Fortin, M., Marsolais, G., 1998. Seroepidemiologic study of Neospora caninum in dairy herds. J. Am. Vet. Med. Assoc. 213, 1595–1598. Sawada, M., Park, C., Kondo, H., Morita, T., Shimada, A., Yamane, I., Umenura, T., 1998. Serological survey of antibody to Neospora caninum in Japanese dogs. J. Vet. Med. Sci. 60, 853–854. Wouda, W., Dijkstra, Th., Kramer, A.M.H., van Maanen, C., Brinkhof, J.M.A., 1999. Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle. Int. J. Parasitol. 29, 1677–1682.