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Models for experimental infection of dogs fed with tissue from fetuses and neonatal cattle naturally infected with Neospora caninum
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Veterinary Parasitology 154 (2008) 151–155 www.elsevier.com/locate/vetpar
Short communication
Models for experimental infection of dogs fed with tissue from fetuses and neonatal cattle naturally infected with Neospora caninum C.J.R. Cedillo a, M.J.J. Martı´nez b, A.M. Santacruz c, R.V.M. Banda d, S.E. Morales e,* a
Instituto Tecnolo´gico de Sonora, Campus Na´inari, Edificio LV-100, Interior CV-113, Oficina LV-118, 5 de Fsebrero 818 Sur, Col. Centro, Ciudad Obrego´n, Sonora, Mexico b Departamento de Medicina Preventiva, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria. Avenida Universidad 3000, Me´xico, D.F. C.P. 04510, Mexico c Centro Agropecuario Industrial de Tizayuca, S.A. (CAITSA), Parque Industrial Tizayuca, Kilo´metro 51.5 carretera libre Me´xico–Pachuca, Hidalgo, Mexico d Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP) del Centro Nacional de Investigaciones en Microbiologı´a Veterinaria (CENID, Microbiologı´a). Carretera Me´xico Toluca Km 15.5, Me´xico, D.F., Mexico e Departamento de Patologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico. Ciudad Universitaria. Avenida Universidad 3000, Me´xico, D.F. C.P. 04510, Mexico Received 7 November 2007; received in revised form 8 February 2008; accepted 21 February 2008
Abstract Three models were designed to investigate the development and enteroepithelial phase of Neospora caninum in dogs, and to induce oocyst production by the parasite. In the first model, three dogs were fed raw fetal bovine tissue on two occasions. The bovine fetal tissue had been stored at 4 8C for 14 days and 16 days, respectively, and had tested positive for neosporosis using histopathology and immunohistochemistry. In the second model, nine dogs from the beginning of the experiment until euthanasia were fed portions of tissue from bovine fetuses that been stored at 4 8C for between 1 and 2 days. Three fetuses had tested positive for neosporosis using histopathology and immunohistochemistry. In the third model, three dogs were fed pieces of raw tissue from two neonatal calves that had not received colostrum, and which had tested positive for antibodies against N. caninum. The brains of these calves were positive for neosporosis by histopathology and immunohistochemistry. In all three models, none of the dogs excreted oocysts of N. caninum, developed intestinal parasites or seroconverted. # 2008 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Dogs; Models of infection; Cattle; Calves; Fetuses
1. Introduction Neosporosis is a major cause of abortion in cattle worldwide, and is also a cause of neuromuscular disease in dogs (Lindsay and Dubey, 2000; Dubey and Schares,
* Corresponding author. Tel.: +55 56 22 58 88/56 16 67 95. E-mail address: [email protected] (S.E. Morales). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.02.025
2006). Experimental studies have indicated that dogs fed on diverse tissues from infected animals, may shed N. caninum oocysts (McAllister et al., 1998; Lindsay et al., 1999; Dijkstra et al., 2001; Gondim et al., 2002; Rodrigues et al., 2004), but whether they can be infected by the ingestion of oocysts is unknown. Although several epidemiological studies have indicated that the presence of dogs on farms may be a risk factor for bovine neosporosis (Bartels et al., 1999; Dijkstra et al.,
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2002; Sanchez et al., 2003), few reports have described the identification of oocysts in the feces of naturally infected dogs (Basso et al., 2001; Slapeta et al., 2002; McGarry et al., 2003; Schares et al., 2005). Therefore, consumption of aborted cattle fetuses would appear not to be an important natural source of infection with N. caninum for dogs (Bergeron et al., 2001). The enteroepithelial phases of this parasite in dogs have not been defined, and likewise very little is known of the frequency of elimination and the resistance of oocysts outside the host (Lindsay et al., 1999; McGarry et al., 2003; Gondim et al., 2005). Three models of experimental infection were developed in this study by feeding dogs fetal and neonatal tissues from cattle naturally infected by the parasite. The aim was to induce the development of the N. caninum and stimulate the production of oocysts, as well as describing the enteroepithelial phase of the parasite in dogs. 2. Materials and methods The fetal and neonatal tissues from cattle used in the three models were collected from the Complejo Agropecuario Industrial de Tizayuca, Sociedad Ano´nima (CAITSA), located at Kilometre 51.5 on the free road Mexico–Pachuca, State of Hidalgo, Mexico. The dogs were housed in the National Institute of Forestry, Agricultural and Veterinary Research (INIFAP), at the National Center for Research in Veterinary Microbiology (CENID, Microbiology) situated on the Mexico Toluca highway at Km 15.5, D.F., Mexico. Laboratory studies were carried out at the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico (UNAM). 2.1. First model Forty aborted fetuses were collected from 22 herds of cattle at CAITSA between August and November, 2004. The gestational age of the fetuses was between 7 and 8 months and they had been aborted not more than 7 days prior to collection. From the time the fetuses were found and collected until processing they were maintained at 4 8C. Necropsy was carried out in order to obtain the brain, heart, skeletal muscle and liver. A sample of each of these tissues was fixed in 10% buffered formalin and processed routinely for histology. The remainder of the tissues was conserved at 4 8C pending confirmation of parasites and/or lesions compatible with neosporosis by histopathology (Dubey and Schares, 2006). Stored tissues of fetuses considered positive were transported
to the INIFAP, CENID, Microbiology, to be administered as food to the dogs. To identify the presence and approximate number of parasites, brain samples manifesting lesions compatible with neosporosis were examined by immunohistochemistry (IHC) using a primary N. caninum antibody of goat origin diluted at 1:1000 (VMRD, Inc., P.O. Box 502 Pullman, WA 99163, USA), following the technique of Morales et al. (2001). For this model, three healthy mixed-breed littermates, one female and two males, were used. The dogs were 1 month of age at the beginning of conditioning. Hematological and biochemical parameters were measured in order to eliminate the possibility of any subclinical abnormality. Anthelmintic (Panacur plus1, Intervet, and Lopatol 1001, Novartis) and anticoccidial (Baycox 5%1, Bayer) treatments were administered. A blood sample was also taken in order to assay antibodies against N. caninum using an enzyme-linked immunosorbent assay (ELISA) (serological detection of specific antibodies to N. caninum by the blocking ELISA method. Institut Pourquier, Version: P00510/01. 326 rue de la Galera 34090 Montpellier, France). The dogs were isolated in individual cages and were fed a commercial diet from 1 to 11 months of age, when study of this model was concluded. In addition, from the beginning of confinement until the model was concluded, the dogs were provided with chunks of raw chicken with the aim of encouraging consumption of fetal tissue. When the dogs were 3 months old, each was fed on two occasions, at an interval of 45 days, with approximately 500 g of a mixture of raw bovine fetal tissues, which had been stored at 4 8C for 14 days and 16 days, respectively, and were considered positive by histopathology and IHC. Forty-five days after consumption, serology was again carried out on the dogs using the same ELISA method. From the second day after consumption of bovine fetal tissues, all the feces from each dog were collected once daily, mixed and 3 g were examined daily, until the conclusion of the model, using flotation in modified Sheather’s sugar solution (s.g. 1.30). Fecal samples were stored at 4 8C until analyzed. These dogs were not euthanatized. 2.2. Second model Forty-two aborted fetuses were collected from cattle in 30 CAITSA herds including the 22 herds in model 1, between December 2004 and June 2005. Fetuses had a gestational age of between 7 and 8 months and not more than 7 days had passed since they had been aborted.
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From the time the fetuses were collected until their processing, they were maintained at 4 8C. Necropsy was carried out on each fetus, and the same organs were obtained for the histopathological and IHC study, using the same techniques as in model 1. The remainder of these tissues was conserved at 4 8C, and was transported immediately to the CENID, Microbiology in order to be administered as food to the dogs. Nine healthy mixed-breed littermate dogs were used, five males and four females. They were divided into three groups of three puppies, which at the beginning of their conditioning were 2, 5 and 7 months of age, respectively. Laboratory analyses and management of the dogs were the same as for model 1. From 3, 6 and 8 months of age, until euthanasia at 8, 11 and 13 months of age, the dogs were fed approximately 800 g of fetal tissue daily. The tissue had been aborted 1 or 2 days previously, and was fed without awaiting the histopathological or IHC results. Serological follow-up was carried out 45 days after the first consumption of fetal tissues and before euthanasia, using the ELISA method described above. The procedures for the collection and evaluation of canine feces were equal as in model 1 from the second day until the time of euthanasia. When the dogs reached 8, 11 and 13 months of age, they were euthanatized by intravenous overdose of sodium pentobarbital (Anestesal1, Pfizer) using 120 mg for each kg bodyweight. A necropsy was carried out and samples were taken from all parts of the intestine; these were fixed in 10% formalin, processed routinely for histology, and examined using an optical microscope. Identical intestinal sections were processed using IHC following the technique described above. 2.3. Third model Two recently born pre-colostrum calves from two of the CAITSA herds were selected, which tested positive for N. caninum antibodies in an indirect ELISA test (CIVTEST bovis Neospora. Avda. La Selva, 135 17170 Amer (Girona) Spain). The calves were euthanatized using a concussion pistol and then exsanguinated. Necropsy was performed to obtain the brain, spinal chord, heart and liver. A small sample of each of these organs was fixed in buffered formalin at 10% and processed routinely for histopathological examination. Identical samples of brain tissue were processed using IHC, employing the same technique as in models 1 and 2. A substantial part of the fresh tissues was administered to the dogs as food. Three healthy poodle littermate puppies were used, one male and two females, which were 1-month-old at
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the beginning of their conditioning to be used in the model. Following the same procedures and laboratory analyses as above. The dogs were housed, treated, observed and fed as in the previous models. When the dogs reached 2 months of age they were transported to CAITSA and, approximately 1 h after the death of the calves, each dog was given approximately 1 kg of a mixture of raw tissues from both calves and subsequently returned to solitary confinement in the CENID, Microbiology. From the second day after tissue consumption until euthanasia, all the feces from each dog were collected and analyzed daily as in the previous models. A blood sample was taken from each dog before it was euthanatized in order to detect antibodies to N. caninum, using the same ELISA method as in models 1 and 2. One dog from this group was euthanatized, as in the second model, at 9, 12 and 16 days, respectively, following consumption of the tissues. Necropsy was undertaken and samples were taken from all regions of the intestine. These were fixed in 10% buffered formalin, processed using the same histological technique, and examined using an optical microscope. Identical samples of these tissues were processed using IHC, as previously described. 3. Results In the first model, of the 40 bovine fetuses collected, only 2 (5%) presented lesions compatible with neosporosis (Dubey and Schares, 2006), and the two brains tested positive for the presence of parasites by IHC. The tissues of these two fetuses were ingested by the dogs. In the second model, of the 42 fetuses collected, 3 (7.1%) presented lesions compatible with neosporosis, and the three brains tested positive for the parasites by IHC. A portion of the tissues of these fetuses was consumed by the dogs. In the third model, only scarce foci of microgliosis were observed in the brain of one the calves on histopathology, and a few cysts containing bradizoites in this calf and a few tachyzoites in the brain of the other calf were identified using IHC. In the three models, none of the dogs excreted oocysts with morphology compatible with N. caninum, and none of them developed parasites, as confirmed by IHC (models 2 and 3), or antibodies to N. caninum. 4. Discussion The methodology employed in the first model, particularly the feeding of tissue from fetuses not more than 7 days after collection, because, hypothetically,
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live parasites could be found in these tissues, as reported by Lindsay et al. (1992). Tissue cysts are able to survive for at least 14 days at 4 8C in refrigerated brain homogenates, and also survived in the intact brain of a mouse carcass refrigerated at 4 8C for 7 days. However, in the present study it was assumed that the dogs were probably not infected due to the autolysis of brain tissue. Although the bovine tissues were conserved at 4 8C, they remained in storage for between 14 and 16 days, which could have resulted in loss of viability and infectiousness. In the second model, although the fetal tissues were administered to the dogs only a few hours after collection, and also in greater quantity, some time may have passed prior to their collection resulting in acceleration of autolysis. The results of the models in this study are similar to those obtained by Bergeron et al. (2001) in Canada, who failed to produce experimental infection in dogs administered fetal tissues from cattle that had been stored at 70 8C for between 4 and 28 days in the first experiment and at 4 8C for between 2 and 4 weeks in a second experiment. However, other studies have reported successful infection of dogs, when administered placentas for consumption (Dijkstra et al., 2001, 2002). These studies suggest that the probability that the dog will become infected is increased if they consume placentas rather than fetuses. Another possible reason why the dogs used in the first two models did not become infected was that the infectious dose may have been very low. The histopathological and immunohistochemical results showed that the tissues of the five fetuses that tested positive for neosporosis contained only a few tachyzoites and cysts. The minimum quantity of parasites required to cause infection under natural conditions is unknown. In reports on naturally infected dogs that excrete oocysts of N. caninum, the factors which promote patent infection have not been precisely defined. However, it has been suggested that, under natural conditions, dogs have access to placentas and to a greater number of recently aborted fetuses, which could facilitate canine infection (Bergeron et al., 2001). It is not known whether age, breed, gender, or immunological conditions have any effect on the susceptibility of dogs to acquiring neosporosis (Lindsay et al., 1999). With respect to age, in a study carried out by Gondim et al. (2005), it was observed that the production of oocysts by puppies was greater than in adult dogs when they were fed tissues of cattle infected with the parasite. For this reason, as in the currently study, young dogs have commonly been used to produce models of infection (Bergeron et al., 2001; Gondim et al., 2002; Rodrigues et al., 2004). In experimental
studies carried out by Lindsay et al. (1999, 2001), it was observed that the immunosuppressed dogs are more easily infected. In the third model of the current study, certain variables were controlled in order to reduce the time between extraction of the brains and their consumption by the dogs, although infection was also not achieved in this case. This might be explained in part because although the parasites may have been viable, their number was insufficient to infect the dogs. In this model, the true quantity of tachyzoites and cysts that were consumed by the dogs is unknown. In agreement with a study carried out by Rodrigues et al. (2004), it would appear that the greater the quantity of infected tissues consumed by dogs, the greater the probability of infection. However, according to the results of Gondim et al. (2002), it is easier to infect dogs if they are fed the tissues of calves intentionally infected with known doses of parasites, even if these are stored at 4 8C for 24 h prior to consumption. In addition, the quantity and variety of bovine tissues that were consumed by the dogs in the study carried out by Gondim et al. (2002) was greater (3 kg) than the amount of tissue consumed by the dogs in the third model of the current study (1 kg). In the current study when the dogs were fed raw chicken, N. caninum had not been detected in birds (Costa et al., 2008), therefore the possibility that the dogs became infected by this means was not considered. Until now it is not known if the dogs can develop the enteroepithelial phases of this parasite by the consumption of infected chicken. It may be concluded that, under the conditions of these three models, none of the dogs excreted oocysts of N. caninum, developed intestinal parasites or seroconverted. It is possible that specific conditions, rarely found in nature, are required in order that the cycle of this parasite between cattle and dog can be successfully completed. Acknowledgements We would like to thank Luis Antonio Morales for his support in the processing of histological samples. This project was partially financed by the PAPIIT IN 212603 project as well as by the UNAM. References Basso, W., Venturini, L., Venturini, M.C., Hill, D.E., Kwok, O.C., Shen, S.K., Dubey, J.P., 2001. First isolation of Neospora caninum from the feces of a naturally infected dog. J. Parasitol. 87, 612–618. Bartels, C.J., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum-associated abortion storms in dairy herds
C.J.R. Cedillo et al. / Veterinary Parasitology 154 (2008) 151–155 in The Netherlands (1995 to1997). Theriogenology 52, 247–257. Bergeron, N., Fecteau, G., Villeneuve, A., Girard, C., Pare´, J., 2001. Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum. Vet. Parasitol. 97, 145– 152. Costa, K.S., Santos, S.L., Uzeˆda, R.S., Pinheiro, A.M., Almeida, M.A., Araujo, F.R., McAllister, M.M., Gondim, L.F., 2008. Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum. Int. J. Parasitol. 38, 157–159. Dijkstra, T., Eysker, M., Schares, G., Conraths, F.J., Wouda, W., Barkema, H.W., 2001. Dogs shed Neospora caninum oocysts after ingestion of naturally infected bovine placenta but not after ingestion of colostrum spiked with Neospora caninum tachyzoites. Int. J. Parasitol. 31, 747–752. Dijkstra, T., Barkema, H.W., Eysker, M., Hesselink, J.W., Wouda, W., 2002. Natural transmission routes of Neospora caninum between farm dogs and cattle. Vet. Parasitol. 105, 99–104. Dubey, J.P., Schares, G., 2006. Diagnosis of bovine neosporosis. Vet. Parasitol. 140, 1–34. Gondim, L.F.P., Gao, L., McAllister, M.M., 2002. Improved production of Neospora caninum oocysts, cyclical oral transmission between dogs and cattle, and in vitro isolation from oocysts. J. Parasitol. 88, 1159–1163. Gondim, L.F.P., McAllister, M.M., Gao, L., 2005. Effects of host maturity and prior exposure history on the production of Neospora caninum oocysts by dogs. Vet. Parasitol. 134, 33–39. Lindsay, D.S., Blagburn, B.L., Dubey, J.P., 1992. Factors affecting the survival of Neospora caninum 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.
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Lindsay, D.S., Dubey, J.P., 2000. Canine neosporosis. J. Vet. Parasitol. 14, 1–11. Lindsay, D.S., Ritter, D.M., Brake, D., 2001. Oocyst excretion in dogs fed mouse brains containing tissue cysts of a cloned line of Neospora caninum. J. Parasitol. 87, 909–911. 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. McGarry, J.W., Stockton, C.M., Williams, D.J.L., Trees, A.J., 2003. Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound. J. Parasitol. 89, 628–630. Morales, E., Trigo, F.J., Ibarra, F., Puente, E., Santacruz, M., 2001. Neosporosis in Mexican dairy herds: lesions and immunohistochemical detection of Neospora caninum in fetuses. J. Comp. Pathol. 125, 58–63. Rodrigues, A.A.R., Gennari, S.M., Aguilar, D.M., Sreekumar, C., Hill, D.E., Miska, K.B., Vianna, M.C.B., Dubey, J.P., 2004. Shedding of Neospora caninum oocysts by dogs fed tissues from naturally infected water buffaloes (Bubalus bubalis) from Brazil. Vet. Parasitol. 124, 139–150. Sanchez, G.F., Morales, S.E., Martinez, M.J., Trigo, T.J.F., 2003. Determination and correlation of anti-Neospora caninum antibodies in dogs and cattle from Mexico. Can. J. Vet. Res. 67, 142–145. Schares, G., Pantchev, N., Barutzki, D., Heydorn, A.O., Bauer, C., Conraths, F.J., 2005. Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany. Int. J. Parasitol. 35, 1525–1537. Slapeta, J.R., Modry, D., Kyselova, I., Horejs, R., Lukes, J., Koudela, B., 2002. Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenic approach. Vet. Parasitol. 109, 157–167.
Veterinary Parasitology 154 (2008) 151–155 www.elsevier.com/locate/vetpar
Short communication
Models for experimental infection of dogs fed with tissue from fetuses and neonatal cattle naturally infected with Neospora caninum C.J.R. Cedillo a, M.J.J. Martı´nez b, A.M. Santacruz c, R.V.M. Banda d, S.E. Morales e,* a
Instituto Tecnolo´gico de Sonora, Campus Na´inari, Edificio LV-100, Interior CV-113, Oficina LV-118, 5 de Fsebrero 818 Sur, Col. Centro, Ciudad Obrego´n, Sonora, Mexico b Departamento de Medicina Preventiva, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria. Avenida Universidad 3000, Me´xico, D.F. C.P. 04510, Mexico c Centro Agropecuario Industrial de Tizayuca, S.A. (CAITSA), Parque Industrial Tizayuca, Kilo´metro 51.5 carretera libre Me´xico–Pachuca, Hidalgo, Mexico d Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP) del Centro Nacional de Investigaciones en Microbiologı´a Veterinaria (CENID, Microbiologı´a). Carretera Me´xico Toluca Km 15.5, Me´xico, D.F., Mexico e Departamento de Patologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico. Ciudad Universitaria. Avenida Universidad 3000, Me´xico, D.F. C.P. 04510, Mexico Received 7 November 2007; received in revised form 8 February 2008; accepted 21 February 2008
Abstract Three models were designed to investigate the development and enteroepithelial phase of Neospora caninum in dogs, and to induce oocyst production by the parasite. In the first model, three dogs were fed raw fetal bovine tissue on two occasions. The bovine fetal tissue had been stored at 4 8C for 14 days and 16 days, respectively, and had tested positive for neosporosis using histopathology and immunohistochemistry. In the second model, nine dogs from the beginning of the experiment until euthanasia were fed portions of tissue from bovine fetuses that been stored at 4 8C for between 1 and 2 days. Three fetuses had tested positive for neosporosis using histopathology and immunohistochemistry. In the third model, three dogs were fed pieces of raw tissue from two neonatal calves that had not received colostrum, and which had tested positive for antibodies against N. caninum. The brains of these calves were positive for neosporosis by histopathology and immunohistochemistry. In all three models, none of the dogs excreted oocysts of N. caninum, developed intestinal parasites or seroconverted. # 2008 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Dogs; Models of infection; Cattle; Calves; Fetuses
1. Introduction Neosporosis is a major cause of abortion in cattle worldwide, and is also a cause of neuromuscular disease in dogs (Lindsay and Dubey, 2000; Dubey and Schares,
* Corresponding author. Tel.: +55 56 22 58 88/56 16 67 95. E-mail address: [email protected] (S.E. Morales). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.02.025
2006). Experimental studies have indicated that dogs fed on diverse tissues from infected animals, may shed N. caninum oocysts (McAllister et al., 1998; Lindsay et al., 1999; Dijkstra et al., 2001; Gondim et al., 2002; Rodrigues et al., 2004), but whether they can be infected by the ingestion of oocysts is unknown. Although several epidemiological studies have indicated that the presence of dogs on farms may be a risk factor for bovine neosporosis (Bartels et al., 1999; Dijkstra et al.,
152
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2002; Sanchez et al., 2003), few reports have described the identification of oocysts in the feces of naturally infected dogs (Basso et al., 2001; Slapeta et al., 2002; McGarry et al., 2003; Schares et al., 2005). Therefore, consumption of aborted cattle fetuses would appear not to be an important natural source of infection with N. caninum for dogs (Bergeron et al., 2001). The enteroepithelial phases of this parasite in dogs have not been defined, and likewise very little is known of the frequency of elimination and the resistance of oocysts outside the host (Lindsay et al., 1999; McGarry et al., 2003; Gondim et al., 2005). Three models of experimental infection were developed in this study by feeding dogs fetal and neonatal tissues from cattle naturally infected by the parasite. The aim was to induce the development of the N. caninum and stimulate the production of oocysts, as well as describing the enteroepithelial phase of the parasite in dogs. 2. Materials and methods The fetal and neonatal tissues from cattle used in the three models were collected from the Complejo Agropecuario Industrial de Tizayuca, Sociedad Ano´nima (CAITSA), located at Kilometre 51.5 on the free road Mexico–Pachuca, State of Hidalgo, Mexico. The dogs were housed in the National Institute of Forestry, Agricultural and Veterinary Research (INIFAP), at the National Center for Research in Veterinary Microbiology (CENID, Microbiology) situated on the Mexico Toluca highway at Km 15.5, D.F., Mexico. Laboratory studies were carried out at the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico (UNAM). 2.1. First model Forty aborted fetuses were collected from 22 herds of cattle at CAITSA between August and November, 2004. The gestational age of the fetuses was between 7 and 8 months and they had been aborted not more than 7 days prior to collection. From the time the fetuses were found and collected until processing they were maintained at 4 8C. Necropsy was carried out in order to obtain the brain, heart, skeletal muscle and liver. A sample of each of these tissues was fixed in 10% buffered formalin and processed routinely for histology. The remainder of the tissues was conserved at 4 8C pending confirmation of parasites and/or lesions compatible with neosporosis by histopathology (Dubey and Schares, 2006). Stored tissues of fetuses considered positive were transported
to the INIFAP, CENID, Microbiology, to be administered as food to the dogs. To identify the presence and approximate number of parasites, brain samples manifesting lesions compatible with neosporosis were examined by immunohistochemistry (IHC) using a primary N. caninum antibody of goat origin diluted at 1:1000 (VMRD, Inc., P.O. Box 502 Pullman, WA 99163, USA), following the technique of Morales et al. (2001). For this model, three healthy mixed-breed littermates, one female and two males, were used. The dogs were 1 month of age at the beginning of conditioning. Hematological and biochemical parameters were measured in order to eliminate the possibility of any subclinical abnormality. Anthelmintic (Panacur plus1, Intervet, and Lopatol 1001, Novartis) and anticoccidial (Baycox 5%1, Bayer) treatments were administered. A blood sample was also taken in order to assay antibodies against N. caninum using an enzyme-linked immunosorbent assay (ELISA) (serological detection of specific antibodies to N. caninum by the blocking ELISA method. Institut Pourquier, Version: P00510/01. 326 rue de la Galera 34090 Montpellier, France). The dogs were isolated in individual cages and were fed a commercial diet from 1 to 11 months of age, when study of this model was concluded. In addition, from the beginning of confinement until the model was concluded, the dogs were provided with chunks of raw chicken with the aim of encouraging consumption of fetal tissue. When the dogs were 3 months old, each was fed on two occasions, at an interval of 45 days, with approximately 500 g of a mixture of raw bovine fetal tissues, which had been stored at 4 8C for 14 days and 16 days, respectively, and were considered positive by histopathology and IHC. Forty-five days after consumption, serology was again carried out on the dogs using the same ELISA method. From the second day after consumption of bovine fetal tissues, all the feces from each dog were collected once daily, mixed and 3 g were examined daily, until the conclusion of the model, using flotation in modified Sheather’s sugar solution (s.g. 1.30). Fecal samples were stored at 4 8C until analyzed. These dogs were not euthanatized. 2.2. Second model Forty-two aborted fetuses were collected from cattle in 30 CAITSA herds including the 22 herds in model 1, between December 2004 and June 2005. Fetuses had a gestational age of between 7 and 8 months and not more than 7 days had passed since they had been aborted.
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From the time the fetuses were collected until their processing, they were maintained at 4 8C. Necropsy was carried out on each fetus, and the same organs were obtained for the histopathological and IHC study, using the same techniques as in model 1. The remainder of these tissues was conserved at 4 8C, and was transported immediately to the CENID, Microbiology in order to be administered as food to the dogs. Nine healthy mixed-breed littermate dogs were used, five males and four females. They were divided into three groups of three puppies, which at the beginning of their conditioning were 2, 5 and 7 months of age, respectively. Laboratory analyses and management of the dogs were the same as for model 1. From 3, 6 and 8 months of age, until euthanasia at 8, 11 and 13 months of age, the dogs were fed approximately 800 g of fetal tissue daily. The tissue had been aborted 1 or 2 days previously, and was fed without awaiting the histopathological or IHC results. Serological follow-up was carried out 45 days after the first consumption of fetal tissues and before euthanasia, using the ELISA method described above. The procedures for the collection and evaluation of canine feces were equal as in model 1 from the second day until the time of euthanasia. When the dogs reached 8, 11 and 13 months of age, they were euthanatized by intravenous overdose of sodium pentobarbital (Anestesal1, Pfizer) using 120 mg for each kg bodyweight. A necropsy was carried out and samples were taken from all parts of the intestine; these were fixed in 10% formalin, processed routinely for histology, and examined using an optical microscope. Identical intestinal sections were processed using IHC following the technique described above. 2.3. Third model Two recently born pre-colostrum calves from two of the CAITSA herds were selected, which tested positive for N. caninum antibodies in an indirect ELISA test (CIVTEST bovis Neospora. Avda. La Selva, 135 17170 Amer (Girona) Spain). The calves were euthanatized using a concussion pistol and then exsanguinated. Necropsy was performed to obtain the brain, spinal chord, heart and liver. A small sample of each of these organs was fixed in buffered formalin at 10% and processed routinely for histopathological examination. Identical samples of brain tissue were processed using IHC, employing the same technique as in models 1 and 2. A substantial part of the fresh tissues was administered to the dogs as food. Three healthy poodle littermate puppies were used, one male and two females, which were 1-month-old at
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the beginning of their conditioning to be used in the model. Following the same procedures and laboratory analyses as above. The dogs were housed, treated, observed and fed as in the previous models. When the dogs reached 2 months of age they were transported to CAITSA and, approximately 1 h after the death of the calves, each dog was given approximately 1 kg of a mixture of raw tissues from both calves and subsequently returned to solitary confinement in the CENID, Microbiology. From the second day after tissue consumption until euthanasia, all the feces from each dog were collected and analyzed daily as in the previous models. A blood sample was taken from each dog before it was euthanatized in order to detect antibodies to N. caninum, using the same ELISA method as in models 1 and 2. One dog from this group was euthanatized, as in the second model, at 9, 12 and 16 days, respectively, following consumption of the tissues. Necropsy was undertaken and samples were taken from all regions of the intestine. These were fixed in 10% buffered formalin, processed using the same histological technique, and examined using an optical microscope. Identical samples of these tissues were processed using IHC, as previously described. 3. Results In the first model, of the 40 bovine fetuses collected, only 2 (5%) presented lesions compatible with neosporosis (Dubey and Schares, 2006), and the two brains tested positive for the presence of parasites by IHC. The tissues of these two fetuses were ingested by the dogs. In the second model, of the 42 fetuses collected, 3 (7.1%) presented lesions compatible with neosporosis, and the three brains tested positive for the parasites by IHC. A portion of the tissues of these fetuses was consumed by the dogs. In the third model, only scarce foci of microgliosis were observed in the brain of one the calves on histopathology, and a few cysts containing bradizoites in this calf and a few tachyzoites in the brain of the other calf were identified using IHC. In the three models, none of the dogs excreted oocysts with morphology compatible with N. caninum, and none of them developed parasites, as confirmed by IHC (models 2 and 3), or antibodies to N. caninum. 4. Discussion The methodology employed in the first model, particularly the feeding of tissue from fetuses not more than 7 days after collection, because, hypothetically,
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live parasites could be found in these tissues, as reported by Lindsay et al. (1992). Tissue cysts are able to survive for at least 14 days at 4 8C in refrigerated brain homogenates, and also survived in the intact brain of a mouse carcass refrigerated at 4 8C for 7 days. However, in the present study it was assumed that the dogs were probably not infected due to the autolysis of brain tissue. Although the bovine tissues were conserved at 4 8C, they remained in storage for between 14 and 16 days, which could have resulted in loss of viability and infectiousness. In the second model, although the fetal tissues were administered to the dogs only a few hours after collection, and also in greater quantity, some time may have passed prior to their collection resulting in acceleration of autolysis. The results of the models in this study are similar to those obtained by Bergeron et al. (2001) in Canada, who failed to produce experimental infection in dogs administered fetal tissues from cattle that had been stored at 70 8C for between 4 and 28 days in the first experiment and at 4 8C for between 2 and 4 weeks in a second experiment. However, other studies have reported successful infection of dogs, when administered placentas for consumption (Dijkstra et al., 2001, 2002). These studies suggest that the probability that the dog will become infected is increased if they consume placentas rather than fetuses. Another possible reason why the dogs used in the first two models did not become infected was that the infectious dose may have been very low. The histopathological and immunohistochemical results showed that the tissues of the five fetuses that tested positive for neosporosis contained only a few tachyzoites and cysts. The minimum quantity of parasites required to cause infection under natural conditions is unknown. In reports on naturally infected dogs that excrete oocysts of N. caninum, the factors which promote patent infection have not been precisely defined. However, it has been suggested that, under natural conditions, dogs have access to placentas and to a greater number of recently aborted fetuses, which could facilitate canine infection (Bergeron et al., 2001). It is not known whether age, breed, gender, or immunological conditions have any effect on the susceptibility of dogs to acquiring neosporosis (Lindsay et al., 1999). With respect to age, in a study carried out by Gondim et al. (2005), it was observed that the production of oocysts by puppies was greater than in adult dogs when they were fed tissues of cattle infected with the parasite. For this reason, as in the currently study, young dogs have commonly been used to produce models of infection (Bergeron et al., 2001; Gondim et al., 2002; Rodrigues et al., 2004). In experimental
studies carried out by Lindsay et al. (1999, 2001), it was observed that the immunosuppressed dogs are more easily infected. In the third model of the current study, certain variables were controlled in order to reduce the time between extraction of the brains and their consumption by the dogs, although infection was also not achieved in this case. This might be explained in part because although the parasites may have been viable, their number was insufficient to infect the dogs. In this model, the true quantity of tachyzoites and cysts that were consumed by the dogs is unknown. In agreement with a study carried out by Rodrigues et al. (2004), it would appear that the greater the quantity of infected tissues consumed by dogs, the greater the probability of infection. However, according to the results of Gondim et al. (2002), it is easier to infect dogs if they are fed the tissues of calves intentionally infected with known doses of parasites, even if these are stored at 4 8C for 24 h prior to consumption. In addition, the quantity and variety of bovine tissues that were consumed by the dogs in the study carried out by Gondim et al. (2002) was greater (3 kg) than the amount of tissue consumed by the dogs in the third model of the current study (1 kg). In the current study when the dogs were fed raw chicken, N. caninum had not been detected in birds (Costa et al., 2008), therefore the possibility that the dogs became infected by this means was not considered. Until now it is not known if the dogs can develop the enteroepithelial phases of this parasite by the consumption of infected chicken. It may be concluded that, under the conditions of these three models, none of the dogs excreted oocysts of N. caninum, developed intestinal parasites or seroconverted. It is possible that specific conditions, rarely found in nature, are required in order that the cycle of this parasite between cattle and dog can be successfully completed. Acknowledgements We would like to thank Luis Antonio Morales for his support in the processing of histological samples. This project was partially financed by the PAPIIT IN 212603 project as well as by the UNAM. References Basso, W., Venturini, L., Venturini, M.C., Hill, D.E., Kwok, O.C., Shen, S.K., Dubey, J.P., 2001. First isolation of Neospora caninum from the feces of a naturally infected dog. J. Parasitol. 87, 612–618. Bartels, C.J., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum-associated abortion storms in dairy herds
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