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Histopathological and clinical investigations in Neospora caninum experimentally infected broiler chicken embryonated eggs
Veterinary Parasitology 166 (2009) 185–190
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Histopathological and clinical investigations in Neospora caninum experimentally infected broiler chicken embryonated eggs Maryam Mansourian a,*, Azizollah Khodakaram-Tafti a, Mehdi Namavari b a b
Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz 1731-71345, Iran Razi Vaccine and Serum Research Institute, Shiraz, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2009 Received in revised form 11 September 2009 Accepted 23 September 2009
To date, despite the limited information about the role of birds in Neospora caninum infection, there are no reports regarding the role of broiler chickens. In the present study, an experimental infection with N. caninum NC-1 isolate was conducted in embryonated eggs from 70 Lohman broiler chickens randomly divided into seven equal groups. At 8 days of incubation, six groups were inoculated via chorioallantoic (CA) liquid with different dilutions (10, 102, 103, 104, 105, and 106) of tachyzoites/embryonated egg. The 7th group was considered the control. The mortality rate, gross and histopathologic changes of tissues of the dead embryos and live hatched chickens up to 60 days old were studied and evaluated. There were no hatchings in groups with 105 and 106 tachyzoites. In groups with 10, 102, 103 and 104 tachyzoites there were 8, 7, 5 and 2 hatchings, respectively. From the surviving chickens only one 104 tachyzoites inoculated chicken showed clinical neurologic signs. In all groups the main gross lesions were hemorrhage associated with thickening of the CA membranes. Three chickens (one chicken with 104 tachyzoites and two with 105 tachyzoites) showed arthritis in the feet joints after two weeks of inoculation. Microscopic examination of the heart, liver and chorioallantoic membrane revealed acute neosporosis and, in some cases, granulomatous inflammation. Our findings implied that broiler chicken embryonated egg is a completely suitable animal model for biological studies of acute neosporosis studies and genetic susceptibility can be propounded in different chicken breeds. It seems 103 tachyzoites dilution is equal to LD50 in N. caninum infection in broiler chicken embryonated eggs and can be used in other experimental investigations. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Neospora caninum Broiler chicken Embryonated egg Experimental infection
1. Introduction Neospora caninum is a heterogeneous cyst-forming coccidian parasite that has emerged as an important cause of reproductive failure in cattle worldwide (Dubey, 2003). Since its introduction in the 1980s (Bjerka˚s et al., 1984), an extensive number of species have been investigated for their role in the parasite’s life-cycle. N. caninum is highly pathogenic for cows and dogs and could also induce disease in sheep, goats, horses and deer (Georgieva et al., 2006).
* Corresponding author. Tel.: +98 711 228 6940; fax: +98 711 228 6950. E-mail address: [email protected] (M. Mansourian). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.09.041
High seroprevalence to N. caninum was found in heifers/ steers and adults in cattle raised on farms having a large number of dogs, and in cattle raised in buffer zones having high minimum temperatures in the spring, and a narrow extension of summer normalized difference vegetation index (Rinaldi et al., 2005). Moreover, the presence of birds in cattle raising farms represents increased risk factors for abortion storms related to N. caninum (Bartels et al., 1999). It is known that canids prey upon bird and could be a good source of infection to the parasite’s definitive hosts (McGuire et al., 1999; Gondim, 2006). Among birds, pigeons and zebra finches were shown to be potential intermediate hosts of the parasite, since experimental infections have established that birds may be susceptible to infection by the protozoan (McGuire et al., 1999). It is
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M. Mansourian et al. / Veterinary Parasitology 166 (2009) 185–190
known that chickens (Gallus domesticus) are natural intermediate hosts of N. caninum (Costa et al., 2008). Pigeons (Columba livia) as a suitable experimental model for N. caninum infection in birds were also recommended (Mineo et al., 2009). Furuta et al. (2007) demonstrated that chickens may be intermediate hosts of N. caninum and embryonated eggs could also be a useful model to study the parasite’s biology. There is limited information concerning the role of birds in N. caninum infection (McGuire et al., 1999; Gondim, 2006; Costa et al., 2008; Mineo et al., 2009; Furuta et al., 2007); but nothing has been written about the role of broiler chickens. Therefore, this study was conducted to evaluate clinical and histopathologic signs of experimentally infected broiler chicken embryonated eggs with N. caninum. 2. Materials and methods 2.1. Parasites In the present study, the N. caninum NC-1 isolate (Dubey et al., 1988) was used for experimental infections. According to the Furuta et al. (2007) method, the parasites were cultured in Vero cell line with fresh RPMI medium (Sigma Co., USA) and 2% fetal calf serum. RPMI medium was supplemented with an antibiotic/antimycotic solution, containing penicillin (10,000 U), streptomycin (100 mg), and amphotericin B (25 mg) (Invitrogen, USA), then harvested by scraping off the cell monolayer about 4– 5 days after infection. For preparation of the necessary inoculum doses, parasites were pelleted by centrifugation, concentration set with a Neubauer chamber, and diluted in RPMI medium. 2.2. Animals and experimental infections Experimental infections were conducted in embryonated eggs from 70 Lohman broiler chickens randomly divided into seven equal groups. All eggs were maintained in an incubator with a controlled temperature, humidity and rotation until 8 days of incubation. At this time six groups were inoculated with different dilutions (10, 102, 103, 104, 105, and 106) of tachyzoites/embryonated egg. The 7th group was considered the control and was inoculated with a sterile cell culture medium. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs (Warren and Russ, 1948). The embryos were observed twice daily until hatching, and any mortality was studied. Dead embryos were necropsied and suitable samples from different tissues including the liver, heart and brain were collected for histopathological study. The samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned at about 5 mm, stained with hematoxylin and eosin and studied microscopically. Live chickens after hatch were also observed daily and any clinical signs recorded. Chickens without advanced clinical signs or apparently normal were followed for two months to note any clinical signs or pathologic lesions. After two months the live chickens were euthanized and then the dead or euthanized chickens
were necropsied and suitable samples from the mentioned tissues were collected and examined. 2.3. Statistical analysis The LD50 was calculated by the Reed and Muench method (Pizzi, 1950). 3. Results 3.1. Mortality rate of experimental infection As are shown in Table 1 and Fig. 1, the inoculated experiment groups showed mortality between 18 and 21 days of incubation, but two groups (with 105 and 106 tachyzoites) showed mortality about 4 days after inoculation. In the group with 106 tachyzoites, six embryos 5 days post-infection (P.I.), three, 6 days P.I., and one, on the 7th day P.I. died. In the group with 105 tachyzoites, one embryo 5 days P.I., three, 6 days P.I., and six, on the 7th, 8th and 9th days P.I. (two chickens per day) died. In the group with 104 tachyzoites, five embryos 9 days P.I., two, 10 days P.I., and one, on the 11th day P.I. died. In the group with 103 tachyzoites, three embryos 10 days P.I. and two, 11 days P.I. died. In the group with 102 tachyzoites, two embryos 11 days P.I. and one, 12 days P.I. died. In the group with 10 tachyzoites, just two embryos died 12 days P.I. In the groups with 10, 102, 103 and 104 tachyzoites there were 8, 7, 5 and 2 hatchings, respectively. 3.1.1. Postmortem findings Gross examination of dead embryos showed mainly hemorrhages associated with the thickening of CA membranes. There were no macroscopic lesions in the heart, liver and brain of the dead embryos in any of the groups, but the CA membrane presented gross lesions in dead embryos in the groups with 104, 105, and 106 inoculated doses (Fig. 2). In gross examination of the eggs of the groups with 10, 102, 103 and104 tachyzoites, hemorrhage was seen at the inner surface of the egg shells. In groups with 10 and 102 tachyzoites, the hemorrhages were mild, while in the group with 103 tachyzoites they were moderate, and in the group with 104 tachyzoites the hemorrhages were severe. In the chickens the CA membrane was not absorbed completely after hatching in all four groups with 10, 102, 103 and 104 tachyzoites, and in all groups with 103 and 104 tachyzoites, a little hemorrhage at the site of the junction between the CA membrane and the chicken was seen. 3.1.2. Clinical signs after hatching After hatching, only one surviving chicken (with 104 tachyzoites inoculum dose) showed neurologic signs. Lack of coordination, pedalling movements, hind limb paralysis, and circular walking patterns were observed in this chicken (Fig. 3). Three chickens (one chicken with 104 tachyzoites and two with 105 tachyzoites) showed arthritis in the feet joints after two weeks of inoculation. There was swelling, redness and ulceration in the surfaces of those joints (Fig. 4).
M. Mansourian et al. / Veterinary Parasitology 166 (2009) 185–190
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Table 1 Pathologic effects and clinical signs in Neospora caninum experimentally infected broiler chicken embryonated eggs. (+: mild lesion; ++: moderate lesion; +++: severe lesion). Inoculum dose
Number of hatched chicken
Mortality rate until 60 days after hatch
Neurologic signs
Arthritis
Severity of microscopic lesion
10 102 103 104 105 106
8 7 5 2 0 0
1 1 2 2 0 0
– – – 1 – –
0 0 0 1 2 0
+ + ++ +++ +++ +++
3.1.3. Histopathologic findings Microscopic examination of the liver, heart, brain and CA membrane revealed acute neosporosis. Hemorrhage, necrosis and infiltration of the mononuclear inflammatory cell and tachyzoites aggregations were seen in the CA membrane, heart, liver and brain (Fig. 5). In groups with the higher inoculum dose (groups with more than 103 tachyzoites) the lesions became more severe and peracute. In the cases with 103 and 104 tachyzoites granulomatous inflammation was seen with aggregation of numerous macrophages in the CA membranes (Fig. 6), liver (Fig. 7) and between the myocardial cells of the hearts of the chickens (Fig. 8). Microscopic examination of the affected joints revealed severe arthritis. There was a chronic active inflammation reaction and also numerous
macrophages infiltrated the synovial membrane associated with heterophils.
Fig. 1. Mortality rate in embryonated eggs experimentally infected with Neospora caninum tachyzoites, with different inoculum doses.
Fig. 3. Lack of coordination, pedalling movements or hind limb paralysis, and circular walking patterns in infected broiler chicken with 104 N. caninum tachyzoites.
Fig. 2. Neospora caninum experimentally infected broiler chicken embryo. Note macroscopic lesion in CA membrane of 18-day-old embryo experimentally infected with 106 Neospora caninum NC-1 tachyzoites (arrow).
3.2. LD50 calculation According to the mortality rates that were summarized in Table 1 and the calculations done using the Reed and
Fig. 4. Swelling and deformity in joints of experimentally infected broiler chicken with 104 N. caninum tachyzoites.
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Muench method (Pizzi, 1950), LD50 was shown to be between 102 and 103 doses and about 102.3 tachyzoites were administered in the chorioallantoic (CA) liquid of the embryonated eggs in 0.1 ml volume per egg. 4. Discussion
Fig. 5. (A) Granulomatous meningitis. Mononuclear cell infiltration in the surface of the cerebellum (H&E 100). (B) Higher magnification of granulomatous inflammation in cerebellum (H&E 400).
Fig. 6. Granulomatous inflammation in CA membrane. Numerous macrophage cells have been infiltrated in CA membrane (H&E 100).
Embryonated eggs have been used as a model for protozoan isolation, propagation, and parasite biology studies (Wunderlin et al., 1997; Que et al., 2004; Furuta et al., 2007). The availability and affordability of embryonated eggs make them good models for experimental studies. Also, embryonated eggs may serve as a valuable tool to study different parasite stages, as seen with Trypanosoma cruzi, which displayed vertebrate and invertebrate specific stages inside egg yolks and CA membranes (Mello and Deane, 1976). To date, there is only one report about experimental N. caninum infection in embryonated eggs (Furuta et al., 2007) and there has been no investigation into experimental N. caninum infections in broiler embryonated eggs. In the present study, neurologic signs like ataxia, lack of coordination, pedalling movements or hind limb paralysis and circular walking patterns in just one chicken from the group with 104 tachyzoites was seen, whereas in the Furuta et al. (2007) study, only 9 egg-laying chickens that hatched their shells (from groups with 105 and 106) present clinical alterations due to infection. Gross examination of the eggs of the groups with 10, 102, 103 and 104 tachyzoites showed that the hemorrhages at the inner surface of the shells of the eggs in the groups with 10 and 102 tachyzoites were mild; in the group with 103 tachyzoites were moderate, and in the group with 104 tachyzoites were severe. These results showed that the severity of the lesions were dependent on the inoculum dose. Furuta et al. (2007) revealed that N. caninum had more lesions in embryos which received higher amounts of tachyzoites in egg-laying chicken embryonated eggs, which is in accordance with our findings. The mortality rate was completely dependent on the inoculum dose as well. In the groups with 10, 102, 103 and 104 tachyzoites; there were 8, 7, 5 and 2 hatches, respectively, but there were no hatchings in groups with 105 and 106 tachyzoites. But Furuta et al. (2007), with the inoculation of different doses of 103 to 104 N. caninum tachyzoites in egg-laying chicken embryonated eggs, showed that mortality rate was not related to inoculum dose, since all groups presented similar death patterns recorded after 7 days of infection. Therefore, in contrast to the egg-laying chickens, our findings showed that lesions and the mortality rate in broiler chickens that were inoculated with different doses of 10 to 106 N. caninum tachyzoites were completely dependent on the inoculum dose. The microscopic findings of our study in broiler chickens showed that histopathologic lesions of acute neosporosis were dependent on the inoculum dose in the liver, heart, CA membrane and brain. Hemorrhage, necrosis and infiltration of the mononuclear inflammatory cell and tachyzoites aggregations were seen in the CA membrane, heart, liver and brain, and in groups with a higher
M. Mansourian et al. / Veterinary Parasitology 166 (2009) 185–190
Fig. 7. Granulomatous hepatitis. Centrilobular mononuclear cell (macrophages higher in number) infiltration in liver (H&E 400).
189
Three chickens (one chicken with 104 tachyzoites and two with 105 tachyzoites) showed arthritis after two weeks of inoculation. Microscopic examination of the affected joints revealed severe arthritis. There was a chronic active inflammation reaction and also numerous macrophages infiltrated the synovial membrane associated with heterophils. The presence of heterophils might be due to acute reaction to synovial injury or a secondary infection. In seeking to develop a laboratory animal model for neosporosis, scientists have found that inbred mouse strains vary in their susceptibility. Most mouse strains are resistant, but BALB/c and C57BL/6 mice develop clinical neosporosis and meningoencephalitis after inoculation with the parasite (Lindsay et al., 1995; Long et al., 1998). Genetic susceptibility was also shown in bovine species (Pan et al., 2004). Therefore, in accordance with recent investigations, our findings imply that genetic susceptibility can be propounded in different breeds of chickens. In conclusion, our findings imply that genetic susceptibility can be propounded in different breeds of chickens and broiler chicken embryonated egg is a completely suitable animal model for acute neosporosis. LD50 was determined to be between 102 and 103 doses and about 102.3 tachyzoites which were administered in the chorioallantoic (CA) liquid of the embryonated eggs in 0.1 ml volume per egg in N. caninum infection in broiler chicken embryonated eggs that can be used in other experimental investigations. References
Fig. 8. Granulomatous myocarditis in Neospora caninum experimentally infected broiler chicken embryo. Numerous macrophages have been infiltrated in heart (H&E 400).
inoculum dose (groups with more than 103 tachyzoites) lesions became more severe and peracute. In accordance with our findings, in the Furuta et al. (2007) study, in the embryos that received 105 and 106 tachyzoites, intense parasite multiplication produced extensive inflammation throughout the CA membranes, followed by widespread necrosis and the marked presence of parasitophagous vacuoles in tissues, especially in the brain, heart and liver. In cases with 103 and 104 tachyzoites; granulomatous inflammation was seen with aggregation of numerous macrophages in the CA membranes, liver and between myocardial cells of the hearts of the chickens. Corbellini et al. (2001) showed similar lesions (granulomatous encephalitis) in the brain of a goat kid, and Lindsay et al. (1996) showed granulomatous inflammation in the central nervous system of a foal.
Bartels, C.J.M., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995–1997). Theriogenology 52, 247–257. Bjerka˚s, I., Mohn, S.F., Presthus, J., 1984. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Zeitschrift fu¨r Parasitenkunde 70, 271–274. Corbellini, L.G., Colodel, E.M., Driemeier, D., 2001. Granulomatous encephalitis in a neurologically impaired goat kid associated with degeneration of Neospora caninum tissue cysts. J. Vet. Diagn. Invest. 13, 416– 419. Costa, K.S., Santos, S.L., Uzeˆda, R.S., Pinheiro, A.M., Almeida, M.A.O., Arau´jo, F.R., McAllister, M.M., Gondim, L.F.P., 2008. Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum. Int. J. Parasitol. 38, 157–159. Dubey, J.P., 2003. Neosporosis in cattle. J. Parasitol. 89, S42–S56. Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J., Uggla, A., 1988. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192, 1269–1285. Furuta, P.I., Mineo, T.W.P., Carrasco, A.O.T., Godoy, G.S., Pinto, A.A., Machado, R.Z., 2007. Neospora caninum infection in birds: experimental infections in chicken and embryonated eggs. Parasitology 1–9. Georgieva, D.A., Prelezov, P.N., Koinarski, V.T.S., 2006. Neospora caninum and neosporosis in animals – a review. Bulgar. J. Vet. Med. 9 (1), 1–26. Gondim, L.F., 2006. Neospora caninum in wildlife. Trends Parasitol. 22, 247–252. Lindsay, D.S., Lenz, S.D., Cole, R.A., Dubey, J.P., Blagburn, B.L., 1995. Mouse model for central nervous system Neospora caninum infections. J. Parasitol. 81, 313–315. Lindsay, D.S., Steinberg, H., Dubielzig, R.R., 1996. Central nervous system neosporosis in a foal. J. Vet. Diagn. Invest. 8, 507–510. Long, M.T., Baszler, T.V., Mathison, B.A., 1998. Comparison of intracerebral parasite load, lesion development, and systemic cytokines in mouse strains infected with Neospora caninum. J. Parasitol. 84, 316–320. McGuire, A.M., McAllister, M.M., Wills, R.A., Tranas, J.D., 1999. Experimental inoculation of domestic pigeons (Columbia livia) and zebra finches (Poephila guttata) with Neospora caninum tachyzoites. Int. J. Parasitol. 29, 1525–1529.
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Mello, M.N., Deane, M.P., 1976. Patterns of development of Trypanosoma cruzi in the embryonated chicken egg. Ann. Trop. Med. Parasit. 70, 380–388. Mineo, T.W.P., Carrasco, A.O.T., Marciano, J.A., Werther, K., Pinto, A.A., Machado, R.Z., 2009. Pigeons (Columba livia) are a suitable experimental model for Neospora caninum infection in birds. Vet. Parasitol. 159, 149–153. Pan, Y., Jansen, G.B., Duffield, T.F., Hietala, S., Kelton, D., Lin, C.Y., Peregrine, A.S., 2004. Genetic susceptibility to Neospora caninum Infection in Holstein cattle in Ontario. J. Dairy Sci. 87, 3967– 3975. Pizzi, M., 1950. Sampling variation of the fifty percent end-point, determined by the Reed–Muench (Behrens) method. Hum. Biol. 22, 151– 190.
Que, X., Wunderlich, A., Joiner, K.A., Reed, S.L., 2004. Toxopain-1 is critical for infection in a novel chicken embryo model of congenital toxoplasmosis. Infect. Immun. 72, 2915–2921. Rinaldi, L., Fusco, G., Musella, V., Veneziano, V., Guarino, A., Taddei, R., Cringoli, G., 2005. Neospora caninum in pastured cattle: determination of climatic, environmental, farm management and individual animal risk factors using remote sensing and geographical information systems. Vet. Parasitol. 128, 219–230. Warren, J., Russ, S.B., 1948. Cultivation of toxoplasma in embryonated eggs – an antigen derived from chorioallantoic membrane. Proc. Soc. Exp. Biol. Med. 67, 85–89. Wunderlin, E., Wild, P., Eckert, J., 1997. Comparative reproduction of Cryptosporidium baileyi in embryonated eggs and in chickens. Parasitol. Res. 83, 712–715.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Histopathological and clinical investigations in Neospora caninum experimentally infected broiler chicken embryonated eggs Maryam Mansourian a,*, Azizollah Khodakaram-Tafti a, Mehdi Namavari b a b
Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz 1731-71345, Iran Razi Vaccine and Serum Research Institute, Shiraz, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2009 Received in revised form 11 September 2009 Accepted 23 September 2009
To date, despite the limited information about the role of birds in Neospora caninum infection, there are no reports regarding the role of broiler chickens. In the present study, an experimental infection with N. caninum NC-1 isolate was conducted in embryonated eggs from 70 Lohman broiler chickens randomly divided into seven equal groups. At 8 days of incubation, six groups were inoculated via chorioallantoic (CA) liquid with different dilutions (10, 102, 103, 104, 105, and 106) of tachyzoites/embryonated egg. The 7th group was considered the control. The mortality rate, gross and histopathologic changes of tissues of the dead embryos and live hatched chickens up to 60 days old were studied and evaluated. There were no hatchings in groups with 105 and 106 tachyzoites. In groups with 10, 102, 103 and 104 tachyzoites there were 8, 7, 5 and 2 hatchings, respectively. From the surviving chickens only one 104 tachyzoites inoculated chicken showed clinical neurologic signs. In all groups the main gross lesions were hemorrhage associated with thickening of the CA membranes. Three chickens (one chicken with 104 tachyzoites and two with 105 tachyzoites) showed arthritis in the feet joints after two weeks of inoculation. Microscopic examination of the heart, liver and chorioallantoic membrane revealed acute neosporosis and, in some cases, granulomatous inflammation. Our findings implied that broiler chicken embryonated egg is a completely suitable animal model for biological studies of acute neosporosis studies and genetic susceptibility can be propounded in different chicken breeds. It seems 103 tachyzoites dilution is equal to LD50 in N. caninum infection in broiler chicken embryonated eggs and can be used in other experimental investigations. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Neospora caninum Broiler chicken Embryonated egg Experimental infection
1. Introduction Neospora caninum is a heterogeneous cyst-forming coccidian parasite that has emerged as an important cause of reproductive failure in cattle worldwide (Dubey, 2003). Since its introduction in the 1980s (Bjerka˚s et al., 1984), an extensive number of species have been investigated for their role in the parasite’s life-cycle. N. caninum is highly pathogenic for cows and dogs and could also induce disease in sheep, goats, horses and deer (Georgieva et al., 2006).
* Corresponding author. Tel.: +98 711 228 6940; fax: +98 711 228 6950. E-mail address: [email protected] (M. Mansourian). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.09.041
High seroprevalence to N. caninum was found in heifers/ steers and adults in cattle raised on farms having a large number of dogs, and in cattle raised in buffer zones having high minimum temperatures in the spring, and a narrow extension of summer normalized difference vegetation index (Rinaldi et al., 2005). Moreover, the presence of birds in cattle raising farms represents increased risk factors for abortion storms related to N. caninum (Bartels et al., 1999). It is known that canids prey upon bird and could be a good source of infection to the parasite’s definitive hosts (McGuire et al., 1999; Gondim, 2006). Among birds, pigeons and zebra finches were shown to be potential intermediate hosts of the parasite, since experimental infections have established that birds may be susceptible to infection by the protozoan (McGuire et al., 1999). It is
186
M. Mansourian et al. / Veterinary Parasitology 166 (2009) 185–190
known that chickens (Gallus domesticus) are natural intermediate hosts of N. caninum (Costa et al., 2008). Pigeons (Columba livia) as a suitable experimental model for N. caninum infection in birds were also recommended (Mineo et al., 2009). Furuta et al. (2007) demonstrated that chickens may be intermediate hosts of N. caninum and embryonated eggs could also be a useful model to study the parasite’s biology. There is limited information concerning the role of birds in N. caninum infection (McGuire et al., 1999; Gondim, 2006; Costa et al., 2008; Mineo et al., 2009; Furuta et al., 2007); but nothing has been written about the role of broiler chickens. Therefore, this study was conducted to evaluate clinical and histopathologic signs of experimentally infected broiler chicken embryonated eggs with N. caninum. 2. Materials and methods 2.1. Parasites In the present study, the N. caninum NC-1 isolate (Dubey et al., 1988) was used for experimental infections. According to the Furuta et al. (2007) method, the parasites were cultured in Vero cell line with fresh RPMI medium (Sigma Co., USA) and 2% fetal calf serum. RPMI medium was supplemented with an antibiotic/antimycotic solution, containing penicillin (10,000 U), streptomycin (100 mg), and amphotericin B (25 mg) (Invitrogen, USA), then harvested by scraping off the cell monolayer about 4– 5 days after infection. For preparation of the necessary inoculum doses, parasites were pelleted by centrifugation, concentration set with a Neubauer chamber, and diluted in RPMI medium. 2.2. Animals and experimental infections Experimental infections were conducted in embryonated eggs from 70 Lohman broiler chickens randomly divided into seven equal groups. All eggs were maintained in an incubator with a controlled temperature, humidity and rotation until 8 days of incubation. At this time six groups were inoculated with different dilutions (10, 102, 103, 104, 105, and 106) of tachyzoites/embryonated egg. The 7th group was considered the control and was inoculated with a sterile cell culture medium. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs (Warren and Russ, 1948). The embryos were observed twice daily until hatching, and any mortality was studied. Dead embryos were necropsied and suitable samples from different tissues including the liver, heart and brain were collected for histopathological study. The samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned at about 5 mm, stained with hematoxylin and eosin and studied microscopically. Live chickens after hatch were also observed daily and any clinical signs recorded. Chickens without advanced clinical signs or apparently normal were followed for two months to note any clinical signs or pathologic lesions. After two months the live chickens were euthanized and then the dead or euthanized chickens
were necropsied and suitable samples from the mentioned tissues were collected and examined. 2.3. Statistical analysis The LD50 was calculated by the Reed and Muench method (Pizzi, 1950). 3. Results 3.1. Mortality rate of experimental infection As are shown in Table 1 and Fig. 1, the inoculated experiment groups showed mortality between 18 and 21 days of incubation, but two groups (with 105 and 106 tachyzoites) showed mortality about 4 days after inoculation. In the group with 106 tachyzoites, six embryos 5 days post-infection (P.I.), three, 6 days P.I., and one, on the 7th day P.I. died. In the group with 105 tachyzoites, one embryo 5 days P.I., three, 6 days P.I., and six, on the 7th, 8th and 9th days P.I. (two chickens per day) died. In the group with 104 tachyzoites, five embryos 9 days P.I., two, 10 days P.I., and one, on the 11th day P.I. died. In the group with 103 tachyzoites, three embryos 10 days P.I. and two, 11 days P.I. died. In the group with 102 tachyzoites, two embryos 11 days P.I. and one, 12 days P.I. died. In the group with 10 tachyzoites, just two embryos died 12 days P.I. In the groups with 10, 102, 103 and 104 tachyzoites there were 8, 7, 5 and 2 hatchings, respectively. 3.1.1. Postmortem findings Gross examination of dead embryos showed mainly hemorrhages associated with the thickening of CA membranes. There were no macroscopic lesions in the heart, liver and brain of the dead embryos in any of the groups, but the CA membrane presented gross lesions in dead embryos in the groups with 104, 105, and 106 inoculated doses (Fig. 2). In gross examination of the eggs of the groups with 10, 102, 103 and104 tachyzoites, hemorrhage was seen at the inner surface of the egg shells. In groups with 10 and 102 tachyzoites, the hemorrhages were mild, while in the group with 103 tachyzoites they were moderate, and in the group with 104 tachyzoites the hemorrhages were severe. In the chickens the CA membrane was not absorbed completely after hatching in all four groups with 10, 102, 103 and 104 tachyzoites, and in all groups with 103 and 104 tachyzoites, a little hemorrhage at the site of the junction between the CA membrane and the chicken was seen. 3.1.2. Clinical signs after hatching After hatching, only one surviving chicken (with 104 tachyzoites inoculum dose) showed neurologic signs. Lack of coordination, pedalling movements, hind limb paralysis, and circular walking patterns were observed in this chicken (Fig. 3). Three chickens (one chicken with 104 tachyzoites and two with 105 tachyzoites) showed arthritis in the feet joints after two weeks of inoculation. There was swelling, redness and ulceration in the surfaces of those joints (Fig. 4).
M. Mansourian et al. / Veterinary Parasitology 166 (2009) 185–190
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Table 1 Pathologic effects and clinical signs in Neospora caninum experimentally infected broiler chicken embryonated eggs. (+: mild lesion; ++: moderate lesion; +++: severe lesion). Inoculum dose
Number of hatched chicken
Mortality rate until 60 days after hatch
Neurologic signs
Arthritis
Severity of microscopic lesion
10 102 103 104 105 106
8 7 5 2 0 0
1 1 2 2 0 0
– – – 1 – –
0 0 0 1 2 0
+ + ++ +++ +++ +++
3.1.3. Histopathologic findings Microscopic examination of the liver, heart, brain and CA membrane revealed acute neosporosis. Hemorrhage, necrosis and infiltration of the mononuclear inflammatory cell and tachyzoites aggregations were seen in the CA membrane, heart, liver and brain (Fig. 5). In groups with the higher inoculum dose (groups with more than 103 tachyzoites) the lesions became more severe and peracute. In the cases with 103 and 104 tachyzoites granulomatous inflammation was seen with aggregation of numerous macrophages in the CA membranes (Fig. 6), liver (Fig. 7) and between the myocardial cells of the hearts of the chickens (Fig. 8). Microscopic examination of the affected joints revealed severe arthritis. There was a chronic active inflammation reaction and also numerous
macrophages infiltrated the synovial membrane associated with heterophils.
Fig. 1. Mortality rate in embryonated eggs experimentally infected with Neospora caninum tachyzoites, with different inoculum doses.
Fig. 3. Lack of coordination, pedalling movements or hind limb paralysis, and circular walking patterns in infected broiler chicken with 104 N. caninum tachyzoites.
Fig. 2. Neospora caninum experimentally infected broiler chicken embryo. Note macroscopic lesion in CA membrane of 18-day-old embryo experimentally infected with 106 Neospora caninum NC-1 tachyzoites (arrow).
3.2. LD50 calculation According to the mortality rates that were summarized in Table 1 and the calculations done using the Reed and
Fig. 4. Swelling and deformity in joints of experimentally infected broiler chicken with 104 N. caninum tachyzoites.
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Muench method (Pizzi, 1950), LD50 was shown to be between 102 and 103 doses and about 102.3 tachyzoites were administered in the chorioallantoic (CA) liquid of the embryonated eggs in 0.1 ml volume per egg. 4. Discussion
Fig. 5. (A) Granulomatous meningitis. Mononuclear cell infiltration in the surface of the cerebellum (H&E 100). (B) Higher magnification of granulomatous inflammation in cerebellum (H&E 400).
Fig. 6. Granulomatous inflammation in CA membrane. Numerous macrophage cells have been infiltrated in CA membrane (H&E 100).
Embryonated eggs have been used as a model for protozoan isolation, propagation, and parasite biology studies (Wunderlin et al., 1997; Que et al., 2004; Furuta et al., 2007). The availability and affordability of embryonated eggs make them good models for experimental studies. Also, embryonated eggs may serve as a valuable tool to study different parasite stages, as seen with Trypanosoma cruzi, which displayed vertebrate and invertebrate specific stages inside egg yolks and CA membranes (Mello and Deane, 1976). To date, there is only one report about experimental N. caninum infection in embryonated eggs (Furuta et al., 2007) and there has been no investigation into experimental N. caninum infections in broiler embryonated eggs. In the present study, neurologic signs like ataxia, lack of coordination, pedalling movements or hind limb paralysis and circular walking patterns in just one chicken from the group with 104 tachyzoites was seen, whereas in the Furuta et al. (2007) study, only 9 egg-laying chickens that hatched their shells (from groups with 105 and 106) present clinical alterations due to infection. Gross examination of the eggs of the groups with 10, 102, 103 and 104 tachyzoites showed that the hemorrhages at the inner surface of the shells of the eggs in the groups with 10 and 102 tachyzoites were mild; in the group with 103 tachyzoites were moderate, and in the group with 104 tachyzoites were severe. These results showed that the severity of the lesions were dependent on the inoculum dose. Furuta et al. (2007) revealed that N. caninum had more lesions in embryos which received higher amounts of tachyzoites in egg-laying chicken embryonated eggs, which is in accordance with our findings. The mortality rate was completely dependent on the inoculum dose as well. In the groups with 10, 102, 103 and 104 tachyzoites; there were 8, 7, 5 and 2 hatches, respectively, but there were no hatchings in groups with 105 and 106 tachyzoites. But Furuta et al. (2007), with the inoculation of different doses of 103 to 104 N. caninum tachyzoites in egg-laying chicken embryonated eggs, showed that mortality rate was not related to inoculum dose, since all groups presented similar death patterns recorded after 7 days of infection. Therefore, in contrast to the egg-laying chickens, our findings showed that lesions and the mortality rate in broiler chickens that were inoculated with different doses of 10 to 106 N. caninum tachyzoites were completely dependent on the inoculum dose. The microscopic findings of our study in broiler chickens showed that histopathologic lesions of acute neosporosis were dependent on the inoculum dose in the liver, heart, CA membrane and brain. Hemorrhage, necrosis and infiltration of the mononuclear inflammatory cell and tachyzoites aggregations were seen in the CA membrane, heart, liver and brain, and in groups with a higher
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Fig. 7. Granulomatous hepatitis. Centrilobular mononuclear cell (macrophages higher in number) infiltration in liver (H&E 400).
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Three chickens (one chicken with 104 tachyzoites and two with 105 tachyzoites) showed arthritis after two weeks of inoculation. Microscopic examination of the affected joints revealed severe arthritis. There was a chronic active inflammation reaction and also numerous macrophages infiltrated the synovial membrane associated with heterophils. The presence of heterophils might be due to acute reaction to synovial injury or a secondary infection. In seeking to develop a laboratory animal model for neosporosis, scientists have found that inbred mouse strains vary in their susceptibility. Most mouse strains are resistant, but BALB/c and C57BL/6 mice develop clinical neosporosis and meningoencephalitis after inoculation with the parasite (Lindsay et al., 1995; Long et al., 1998). Genetic susceptibility was also shown in bovine species (Pan et al., 2004). Therefore, in accordance with recent investigations, our findings imply that genetic susceptibility can be propounded in different breeds of chickens. In conclusion, our findings imply that genetic susceptibility can be propounded in different breeds of chickens and broiler chicken embryonated egg is a completely suitable animal model for acute neosporosis. LD50 was determined to be between 102 and 103 doses and about 102.3 tachyzoites which were administered in the chorioallantoic (CA) liquid of the embryonated eggs in 0.1 ml volume per egg in N. caninum infection in broiler chicken embryonated eggs that can be used in other experimental investigations. References
Fig. 8. Granulomatous myocarditis in Neospora caninum experimentally infected broiler chicken embryo. Numerous macrophages have been infiltrated in heart (H&E 400).
inoculum dose (groups with more than 103 tachyzoites) lesions became more severe and peracute. In accordance with our findings, in the Furuta et al. (2007) study, in the embryos that received 105 and 106 tachyzoites, intense parasite multiplication produced extensive inflammation throughout the CA membranes, followed by widespread necrosis and the marked presence of parasitophagous vacuoles in tissues, especially in the brain, heart and liver. In cases with 103 and 104 tachyzoites; granulomatous inflammation was seen with aggregation of numerous macrophages in the CA membranes, liver and between myocardial cells of the hearts of the chickens. Corbellini et al. (2001) showed similar lesions (granulomatous encephalitis) in the brain of a goat kid, and Lindsay et al. (1996) showed granulomatous inflammation in the central nervous system of a foal.
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