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High seroprevalence of Toxoplasma gondii and Neospora caninum in the Common raven (Corvus corax) in the Northeast of Spain
Research in Veterinary Science 93 (2012) 300–302
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
High seroprevalence of Toxoplasma gondii and Neospora caninum in the Common raven (Corvus corax) in the Northeast of Spain R. Molina-López a, O. Cabezón b, M. Pabón c, L. Darwich c,d, E. Obón a, F. Lopez-Gatius e, J.P. Dubey f, S. Almería c,d,⇑ a
Centre de Fauna Salvatge de Torreferrussa, Catalan Wildlife Service, Direcció General del Medi Natural-Forestal Catalana, SA, Santa Perpètua de la Mogoda, Spain Servei d’Ecopatologia de Fauna Salvatge (SEFaS), Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain Departament de Sanitat i Anatomia Animals, Facultat de Medicina Veterinaria, Universitat Autònoma de Barcelona, 08193 Bellatera, Barcelona, Spain d Centre de Recerca en Sanitat Animal (CReSA), Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain e Department of Animal Production, University of Lleida, Escuela Técnica Superior de Ingeniería Agraria, Lleida, Spain f Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA b c
a r t i c l e
i n f o
Article history: Received 29 April 2011 Accepted 12 May 2011
Keywords: Common ravens Seroprevalence Neospora caninum Toxoplasma gondii
a b s t r a c t In recent years, multiple cases of aggressive behavior of Common ravens (Corvus corax) have been reported by farmers in Catalonia (NE Spain), including attacking of newborn animals and consumption of dead foetuses. In the present study, seroprevalence of Toxoplasma gondii and Neospora caninum was determined from 113 legally trapped and released Common ravens. T. gondii antibodies were found in 91 (80.5%; CI 95%:72–87) of 113 sera tested by the modified agglutination test. Antibodies to N. caninum were found in 24 (35.8%; IC 95%: 24.5–48.5) of 67 Common ravens tested by an indirect fluorescence antibody test with titers ranging from 1:50 (n = 18) to P1:100 (n = 6). To the author’s knowledge, this is the first report of the presence of T. gondii and N. caninum antibodies in C. corax. The seroprevalence detected is one of the highest reported worldwide in wild birds, suggesting an important role for this species in the epidemiology of both parasites. Ó 2011 Elsevier Ltd. All rights reserved.
The Common raven (Corvus corax; Corvidae), also known as the Northern raven, is a large, all-black passerine bird in the crow family. Found across the northern hemisphere, it is the most widely distributed of all corvids. Common ravens are extremely versatile and opportunistic in finding sources of nutrition. They are mainly scavengers feeding on carrion, insects, cereal grains, berries, fruit, small animals, and food waste. They are also known to eat the afterbirth of ewes and other large mammals (Boarman and Heinrich, 1999). The Common raven has coexisted with humans for thousands of years and in some areas is so numerous that it is considered a pest. Recently, multiple cases of aggressive behavior of Common ravens have been reported by farmers in Catalonia (NE Spain), including attacking of newborn animals and consumption of dead foetuses in farms. Toxoplasma gondii and Neospora caninum are two closely-related, intra-cellular apicomplexans of worldwide distribution that have been implicated in abortion and reproductive disorders in livestock (Dubey et al., 2007; Dubey, 2010). Toxoplasmosis, caused by T. gondii, affects most species of warm-blooded animals, includ⇑ Corresponding author at: Departament de Sanitat i Anatomia Animals, Facultat de Medicina Veterinaria, Universitat Autònoma de Barcelona, 08193 Bellatera, Barcelona, Spain. E-mail address: [email protected] (S. Almería). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.05.011
ing birds, and is zoonotic (Dubey, 2009). Felids are the only definitive hosts of T. gondii and they excrete environmentally resistant oocysts. Cats are thought to become infected with T. gondii via predation on infected birds and rodents. T. gondii infections are prevalent in many avian species, and it can cause mortality in some species of birds (Dubey, 2002), including the endangered ‘Alala or Hawaiian crow (Corvus hawaiiensis) (Work et al., 2000). Viable T. gondii has been isolated from the American crow (Corvus brachyrhynchus) from the USA (Finlay and Manwell, 1956), Carrion crow (Corvus corone) from Kazakhstan (Pak, 1976) and Slovakia (Catár, 1974), Jackdaw (Corvus monedula) and Rook (Corvus frugilegus) from Czech Republic (Uterák et al., 1992). It is noteworthy, that these studies were conducted several decades ago. N. caninum is considered one of the most important causes of abortion in cattle worldwide (Dubey et al., 2007). Although canids (dogs, coyotes, dingoes) are the definitive hosts for N. caninum many aspects of transmission of this parasite remain unknown. The presence of birds in cattle raising farms has been associated with outbreaks of abortion and considered as a risk factor for N. caninum infection (Ould-Amrouche et al., 1999; Bartels et al., 1999). Recently the domestic chicken (Gallus domesticus) and the sparrow (Passer domesticus) were reported as natural intermediate hosts for N. caninum (Costa et al., 2008; Gondim et al., 2010).
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Investigation of T. gondii and N. caninum exposure in birds could be a useful way to assess environmental contamination with oocysts, since some avian populations feed directly on the ground and are continuously exposed to oocyst ingestion (Gondim et al., 2010). In addition, investigation of T. gondii and N. caninum infection in scavenger wild birds could also assess the possibility of contact with intermediate hosts and the associated risk for public health. We report for the first time T. gondii and N. caninum infections in the C. corax. A total of 115 wild Common ravens were trapped in Catalonia, NE Spain and brought to the rehabilitation center ‘‘Centro de Recuperación de Fauna Salvaje (CRFS) of Torreferrussa (Santa Perpetua de Moguda, Barcelona, Spain) between 2006 and 2010. The birds were captured with government license after farmers complained of attacks to their animals, in particular neonates and dead foetuses. The species is protected by Spanish national laws and therefore only blood samples were collected by venipuncture of right jugular vein from trapped ravens, in compliance with the Ethical Principles in Animal Research of the CRFS of Torreferrussa. All crows were apparently healthy and were released after sampling. Only two Common ravens died after trapping and brain tissues were collected from the dead birds for detection of the parasite DNA. Sera from these two animals were not available for analysis. Data were collected regarding location and year of sampling. Age was determined whenever possible by external characters, with 94.4% of the birds (67 of 71 birds with age available) being juveniles (61 calendar year). Blood was placed in a serum collection tube until clotted and then centrifuged. Sera were stored at 20 °C until analyzed. Antibodies to T. gondii were determined by the modified agglutination test (MAT) using 2-mercaptoethanol and formalin-fixed whole tachyzoites as described previously (Dubey and Desmonts, 1987). Sera were tested at 1:25, 1:50, 1:100 and 1:500 dilutions. A commercial positive control (Toxotrol-A, Biomerieux, France) diluted from 1:25 to 1:3200 (with a minimum titer of 1:200) as well as negative controls were included in each test. Titers of 1:25 or higher were considered positive and those with doubtful results were re-examined. This technique has been successfully tested in several bird species (Dubey, 2002). Antibodies against N. caninum were assayed using an indirect fluorescent antibody test (IFAT) using slides coated with whole N. caninum tachyzoites (VMRD, Pullman, Washington, USA), and FITC labeled rabbit anti-chicken IgY (IgG) (whole molecule) (SIGMA, Spain) at 1:200 dilutions. This commercially available anti-chicken
IgY antibodies exhibit good cross-reactivity with most avian species (Cray and Villar, 2008) and has been validated for the detection of serum immunoglobulins in wild birds of different species by ELISA (Martínez et al., 2003). A cut-off of 1:50 or higher was considered positive, as previously described for domestic and wildlife species (Ortuño et al., 2002; Almería et al., 2007). Only animals trapped in 2009 and 2010 (n = 67) were available for N. caninum antibody detection. DNA was extracted from approximately 1 g of brain tissue from the two dead Common ravens. DNA was extracted as previously described by Almería et al. (2002). A nested PCR for detection of the repetitive fragment 529 bp (AF487550) of T. gondii was performed as previously described by Su and Dubey (2009). The Nc5 gen PCR was used for N. caninum PCR as described by Liddell et al. (1999) with minor modifications as described by Almería et al. (2002). The seroprevalence to T. gondii and N. caninum was estimated from the ratio of positive to the total number of samples, with binomial confidence intervals of 95%. Associations between the serological results and independent variables such as location and year of sample collection were analyzed using a Pearson’s chi-square test and by Fisher’s exact test when observations/category were <6. Differences between categories were analyzed using the Tukey test. Differences were considered statistically significant when P < 0.05. Statistical analyses were performed using SPSS 15.0 (Statistical Package for Social Sciences (SPSS) Inc.). Antibodies (MAT P 1:25) to T. gondii were found in 91 (80.5%; CI95%: 72.1–87.4) from 113 Common ravens sera with titers of 1:25 in 5 (5.5%), 1:50 in 22 (24.2%), 1:100 in 23 (25.3%), and P1:500 in 41 (45.1%). No statistically significant differences were found between prevalence of infection and age or location of trapped ravens. Statistically significant differences were observed among years of sampling, with the highest seroprevalence in the most recent years (2009–2010) compared to the 2005–2008 period (p = 0.007) (Table 1). N. caninum antibodies were detected by IFAT P 1:50 in 24 of the 67 (35.8%; IC95%: 24.5–48.5) sera analyzed. No statistically significant differences were found between prevalence of infection and age, location, and year of sampling (2009 and 2010) although a trend towards statistically significant differences was observed between years of sampling (p = 0.12) (Table 1). In the samples in which both pathogens were analyzed (n = 67), 20 samples were simultaneously positive to both protozoa, 42 were only positive to T. gondii and 4 were only positive to N. caninum. Only one sample was negative to both protozoa. A trend to-
Table 1 Seroprevalence of Toxoplasma gondii (MAT P 1:25) and Neospora caninum (IFAT P 1:50) in Common ravens (Corvus corax) from Catalonia (NE Spain). Factor
Total Age Location
Year
Category
61 year >1 year Centelles S.M. Centelles Oris Rupit i Pruit 2005 2006 2008 2009 2010
T. gondii (MAT)
N. caninum (IFAT)
Examined (n)
Positive (n)
Prevalence (%)
Examined (n)
Positive (n)
Prevalence (%)
113 67a 4 28 66 2 17 15c 15 14 49 18
91 60 3 22 53 2 12 8 10 9 46 16
80.5 89.5 75.0 78.6 80.3 100.0 70.6 53.3 66.7 64.3 93.9 88.9
67 59b 2 19 38 1 9 ND ND ND 49 18
24 23 0 9 13 0 2
35.8 38.9 0.0 47.4 34.2 0 22.2
15 9
30.6 50.0
MAT: modified agglutination test; IFAT: indirect fluorescent antibody test. a No age data from 42 samples. b No age data from 6 samples. c No data of year of sampling from 2 samples.
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wards a negative association between T. gondii and N. caninum was observed (Fisher exact test, P = 0.052). The presence of T. gondii and N. caninum DNA was not detected in the brain of the only two dead C. corax analyzed by PCR. To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in Common ravens in Spain and in the world. In addition, the prevalence of T. gondii antibodies found in Common ravens in Spain, in a relatively high number of analyzed samples (80.5% of 113 samples), is also one of the highest reported in wild birds worldwide (Dubey, 2002, 2009). Recent studies of T. gondii seroprevalence have shown a high prevalence of T. gondii antibodies in wild birds in Europe but did not examine Common ravens. Aubert et al. (2008) reported prevalence levels in raptors from France, that similar to our study were brought to rehabilitation centers, with levels of 79% of 14 Common buzzards (Buteo buteo), 50% of 12 Tawny owls (Strix aluco) and 11% of 18 Barn owls (Tyto alba). Lopes et al. (2011) reported a prevalence of antibodies in 50% of 52 wild birds brought to rehabilitation centers that include multiple species such as Black kite (Milvus migrans), Booted eagle (Hieraaetus pennatus), Common buzzard, Eurasian sparrowhawk (Accipiter nisus), Northern goshawk (Accipiter gentilis), Eurasian eagle owl (Bubo bubo) and Tawny owl in Portugal. The present results are also the first report of N. caninum antibodies in Common ravens from any country. The presence of birds on dairy farms with neosporosis abortion problems has been considered as a risk factor for N. caninum infection in some studies (Ould-Amrouche et al., 1999; Bartels et al., 1999), but information regarding involvement of wild birds in the epidemiology of N. caninum is scarce, especially in field conditions. The present study showed moderate to high seroprevalence levels of N. caninum antibodies in this species (35.8% of 67 C. corax). The fact that the analyzed ravens in the present study were captured in relation to attacks to farms, could have influenced the high seroprevalence levels observed, but is still a clear indication of the potential role of Common ravens as sentinels for both protozoan infections. A trend towards a negative association between T. gondii and N. caninum was observed. Possible explanations for this result may involve different exposure (with cattle farms being more probable sources of N. caninum, and sheep farms infected with T. gondii) or differences on immunity. Further studies are necessary to confirm this association. An interesting observation was that the highest seroprevalence levels in both T. gondii and N. caninum were observed in the most recent years of study. This could be an indication of an expansion of these protozoan infections in the affected farms over time, although other factors, such as differences in environmental conditions each year, could have also influenced these results. In addition, the prevalence levels observed in the present study corresponded mostly to juvenile Common ravens, since older ravens are difficult to trap. Another explanation could be that in this species, juveniles have a distinct foraging behavior from adults, and tend to group on feeding resources (Dall and Wright, 2009). This may explain high seropositivity levels in the juveniles of this study. Many studies in several species have shown that seroprevalence of T. gondii infection is age-related with higher seroprevalence in older animals (Dubey, 2009), probably due to a greater probability of exposure over time and long persistence of antibodies. Therefore, higher seroprevalence levels might be expected in older Common ravens in the same areas. The present results could indicate a role for Common ravens in the epidemiology of toxoplasmosis and neosporosis. However, detection and/or isolation of the parasite in tissues would be necessary to corroborate that Common ravens are an intermediate host of T. gondii and N. caninum. Unfortunately only two animals could be examined by PCR and both were negative. Further studies
should analyze the presence of T. gondii and N. caninum DNA in tissues and confirm that the Common ravens are intermediate host in the cycle of both parasites. In conclusion, the high seroprevalence levels observed in the present study indicate a high exposure to both T. gondii and N. caninum in Common ravens from Catalonia, Northeast Spain, and suggest an important role for this species in the epidemiology of both parasites. This study received partial support from the Spanish CICYT Grant AGL2007-65521 C02/GAN. The authors thank Dr. Pita Gondim for the generous donation of chicken N. caninum positive control sera for IFAT. We thank all the staff of the Torreferrussa Rehabilitation Center for their devoted care of the patients. References Almería, S., Ferrer, D., Pabón, M., Castellà, J., Mañas, S., 2002. Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum. Veterinary Parasitology 107, 287–294. Almería, S., Vidal, D., Ferrer, D., Pabón, M., Fernández-de-Mera, M.I., Ruiz-Fons, F., Alzaga, V., Marco, I., Calvete, C., Lavin, S., Gortazar, C., López-Gatius, F., Dubey, J.P., 2007. Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain. Veterinary Parasitology 143, 21–28. Aubert, D., Terrier, M.E., Dumètre, A., Barrat, J., Villena, I., 2008. Prevalence of Toxoplasma gondii in raptors from France. Journal of Wildlife Diseases 44, 172– 173. Bartels, C.J., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum associated abortion storms in dairy herds in The Netherlands (1995 to 1997). Theriogenology 52, 247–257. Boarman, W., Heinrich, B., 1999. Corvus corax: Common raven. The Birds of North America 476, 1–32. Catár, G., 1974. Toxoplazmóza vekologickych podmienkach na Slovensku. Biologické Práce (Bratislava) 20, 1–138. Costa, K.S., Santos, S.L., Uzêda, R.S., Pinheiro, A.M., Almeida, M.A., Araújo, F.R., McAllister, M.M., Gondim, L.F., 2008. Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum. International Journal of Parasitology 38, 157–159. Cray, C., Villar, D., 2008. Cross-reactivity of anti-chicken IgY antibody with immunoglobulins of exotic avian species. Veterinary Clinical Pathology 37, 328–331. Dall, S.R.X., Wright, J., 2010. Rich pickings near large communal roosts favor ‘gang’ foraging by juvenile Common ravens, Corvus corax. PloS One 4, 7. Dubey, J.P., Desmonts, G., 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterinary Journal 19, 337–339. Dubey, J.P., 2002. A review of toxoplasmosis in wild birds. Veterinary Parasitology 106, 121–153. Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews 20, 323–367. Dubey, J.P., 2009. Toxoplasmosis of Animals and Humans, second ed. CRC Press Boca Raton, FL, pp. 1–313. Finlay, P., Manwell, R.D., 1956. Toxoplasma from the crow, a new natural host. Experimental Parasitology 5, 149–153. Gondim, L.S., Abe-Sandes, K., Uzêda, R.S., Silva, M.S., Santos, S.L., Mota, R.A., Vilela, S.M., Gondim, L.F., 2010. Toxoplasma gondii and Neospora caninum in sparrows (Passer domesticus) in the Northeast of Brazil. Veterinary Parasitology 168, 121– 124. Liddell, S., Jenkins, M.C., Dubey, J.P., 1999. A competitive PCR assay for quantitative detection of Neospora caninum. International Journal of Parasitology 29, 1583– 1587. Lopes, A.P., Sargo, R., Rodrigues, M., Cardoso, L., 2011. High seroprevalence of antibodies to Toxoplasma gondii in wild animals from Portugal. Parasitology Research 108, 1163–1169. Martínez, J., Tomás, G., Merino, S., Arriero, E., Moreno, J., 2003. Detection of serum immunoglobulins in wild birds by direct ELISA: a methodological study to validate the technique in different species using anti-chicken antibodies. Functional Ecology 17, 700–706. Ortuño, A., Castellà, J., Almería, S., 2002. Seroprevalence of antibodies to Neospora caninum in dogs from Spain. Journal Parasitology 88, 1263–1266. Ould-Amrouche, A., Klein, F., Osdoit, C., Mohammed, H.O., Touratier, A., Sanaa, M., Mialot, J.P., 1999. Estimation of Neospora caninum seroprevalence in dairy cattle from Normandy. France Veterinary Research 30, 531–538. Pak, S.M., 1976. Toxoplasmosis of birds in Kazakhstan. Nauka Publishing, Alma Ata, p. 115. Su, C., Dubey, J.P., 2009. In: Liu, D. (Ed.), Toxoplasma gondii in Molecular Detection of Foodborne Pathogens. CRC Press, Boca Raton, Florida, pp. 741–753. Uterák, I., Hejlicˇek, K., Nezval, J., Folk, C., 1992. Incidence of Toxoplasma gondii in populations of wild birds in the Czech Republic. Avian Pathology 21, 659–665. Work, T.M., Massey, J.G., Rideout, B.A., Gardiner, C.H., Ledig, D.B., Kwok, O.C., Dubey, J.P., 2000. Fatal toxoplasmosis in free-ranging endangered ‘Alala from Hawaii. Journal of Wildlife Diseases 36, 205–212.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
High seroprevalence of Toxoplasma gondii and Neospora caninum in the Common raven (Corvus corax) in the Northeast of Spain R. Molina-López a, O. Cabezón b, M. Pabón c, L. Darwich c,d, E. Obón a, F. Lopez-Gatius e, J.P. Dubey f, S. Almería c,d,⇑ a
Centre de Fauna Salvatge de Torreferrussa, Catalan Wildlife Service, Direcció General del Medi Natural-Forestal Catalana, SA, Santa Perpètua de la Mogoda, Spain Servei d’Ecopatologia de Fauna Salvatge (SEFaS), Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain Departament de Sanitat i Anatomia Animals, Facultat de Medicina Veterinaria, Universitat Autònoma de Barcelona, 08193 Bellatera, Barcelona, Spain d Centre de Recerca en Sanitat Animal (CReSA), Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain e Department of Animal Production, University of Lleida, Escuela Técnica Superior de Ingeniería Agraria, Lleida, Spain f Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA b c
a r t i c l e
i n f o
Article history: Received 29 April 2011 Accepted 12 May 2011
Keywords: Common ravens Seroprevalence Neospora caninum Toxoplasma gondii
a b s t r a c t In recent years, multiple cases of aggressive behavior of Common ravens (Corvus corax) have been reported by farmers in Catalonia (NE Spain), including attacking of newborn animals and consumption of dead foetuses. In the present study, seroprevalence of Toxoplasma gondii and Neospora caninum was determined from 113 legally trapped and released Common ravens. T. gondii antibodies were found in 91 (80.5%; CI 95%:72–87) of 113 sera tested by the modified agglutination test. Antibodies to N. caninum were found in 24 (35.8%; IC 95%: 24.5–48.5) of 67 Common ravens tested by an indirect fluorescence antibody test with titers ranging from 1:50 (n = 18) to P1:100 (n = 6). To the author’s knowledge, this is the first report of the presence of T. gondii and N. caninum antibodies in C. corax. The seroprevalence detected is one of the highest reported worldwide in wild birds, suggesting an important role for this species in the epidemiology of both parasites. Ó 2011 Elsevier Ltd. All rights reserved.
The Common raven (Corvus corax; Corvidae), also known as the Northern raven, is a large, all-black passerine bird in the crow family. Found across the northern hemisphere, it is the most widely distributed of all corvids. Common ravens are extremely versatile and opportunistic in finding sources of nutrition. They are mainly scavengers feeding on carrion, insects, cereal grains, berries, fruit, small animals, and food waste. They are also known to eat the afterbirth of ewes and other large mammals (Boarman and Heinrich, 1999). The Common raven has coexisted with humans for thousands of years and in some areas is so numerous that it is considered a pest. Recently, multiple cases of aggressive behavior of Common ravens have been reported by farmers in Catalonia (NE Spain), including attacking of newborn animals and consumption of dead foetuses in farms. Toxoplasma gondii and Neospora caninum are two closely-related, intra-cellular apicomplexans of worldwide distribution that have been implicated in abortion and reproductive disorders in livestock (Dubey et al., 2007; Dubey, 2010). Toxoplasmosis, caused by T. gondii, affects most species of warm-blooded animals, includ⇑ Corresponding author at: Departament de Sanitat i Anatomia Animals, Facultat de Medicina Veterinaria, Universitat Autònoma de Barcelona, 08193 Bellatera, Barcelona, Spain. E-mail address: [email protected] (S. Almería). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.05.011
ing birds, and is zoonotic (Dubey, 2009). Felids are the only definitive hosts of T. gondii and they excrete environmentally resistant oocysts. Cats are thought to become infected with T. gondii via predation on infected birds and rodents. T. gondii infections are prevalent in many avian species, and it can cause mortality in some species of birds (Dubey, 2002), including the endangered ‘Alala or Hawaiian crow (Corvus hawaiiensis) (Work et al., 2000). Viable T. gondii has been isolated from the American crow (Corvus brachyrhynchus) from the USA (Finlay and Manwell, 1956), Carrion crow (Corvus corone) from Kazakhstan (Pak, 1976) and Slovakia (Catár, 1974), Jackdaw (Corvus monedula) and Rook (Corvus frugilegus) from Czech Republic (Uterák et al., 1992). It is noteworthy, that these studies were conducted several decades ago. N. caninum is considered one of the most important causes of abortion in cattle worldwide (Dubey et al., 2007). Although canids (dogs, coyotes, dingoes) are the definitive hosts for N. caninum many aspects of transmission of this parasite remain unknown. The presence of birds in cattle raising farms has been associated with outbreaks of abortion and considered as a risk factor for N. caninum infection (Ould-Amrouche et al., 1999; Bartels et al., 1999). Recently the domestic chicken (Gallus domesticus) and the sparrow (Passer domesticus) were reported as natural intermediate hosts for N. caninum (Costa et al., 2008; Gondim et al., 2010).
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Investigation of T. gondii and N. caninum exposure in birds could be a useful way to assess environmental contamination with oocysts, since some avian populations feed directly on the ground and are continuously exposed to oocyst ingestion (Gondim et al., 2010). In addition, investigation of T. gondii and N. caninum infection in scavenger wild birds could also assess the possibility of contact with intermediate hosts and the associated risk for public health. We report for the first time T. gondii and N. caninum infections in the C. corax. A total of 115 wild Common ravens were trapped in Catalonia, NE Spain and brought to the rehabilitation center ‘‘Centro de Recuperación de Fauna Salvaje (CRFS) of Torreferrussa (Santa Perpetua de Moguda, Barcelona, Spain) between 2006 and 2010. The birds were captured with government license after farmers complained of attacks to their animals, in particular neonates and dead foetuses. The species is protected by Spanish national laws and therefore only blood samples were collected by venipuncture of right jugular vein from trapped ravens, in compliance with the Ethical Principles in Animal Research of the CRFS of Torreferrussa. All crows were apparently healthy and were released after sampling. Only two Common ravens died after trapping and brain tissues were collected from the dead birds for detection of the parasite DNA. Sera from these two animals were not available for analysis. Data were collected regarding location and year of sampling. Age was determined whenever possible by external characters, with 94.4% of the birds (67 of 71 birds with age available) being juveniles (61 calendar year). Blood was placed in a serum collection tube until clotted and then centrifuged. Sera were stored at 20 °C until analyzed. Antibodies to T. gondii were determined by the modified agglutination test (MAT) using 2-mercaptoethanol and formalin-fixed whole tachyzoites as described previously (Dubey and Desmonts, 1987). Sera were tested at 1:25, 1:50, 1:100 and 1:500 dilutions. A commercial positive control (Toxotrol-A, Biomerieux, France) diluted from 1:25 to 1:3200 (with a minimum titer of 1:200) as well as negative controls were included in each test. Titers of 1:25 or higher were considered positive and those with doubtful results were re-examined. This technique has been successfully tested in several bird species (Dubey, 2002). Antibodies against N. caninum were assayed using an indirect fluorescent antibody test (IFAT) using slides coated with whole N. caninum tachyzoites (VMRD, Pullman, Washington, USA), and FITC labeled rabbit anti-chicken IgY (IgG) (whole molecule) (SIGMA, Spain) at 1:200 dilutions. This commercially available anti-chicken
IgY antibodies exhibit good cross-reactivity with most avian species (Cray and Villar, 2008) and has been validated for the detection of serum immunoglobulins in wild birds of different species by ELISA (Martínez et al., 2003). A cut-off of 1:50 or higher was considered positive, as previously described for domestic and wildlife species (Ortuño et al., 2002; Almería et al., 2007). Only animals trapped in 2009 and 2010 (n = 67) were available for N. caninum antibody detection. DNA was extracted from approximately 1 g of brain tissue from the two dead Common ravens. DNA was extracted as previously described by Almería et al. (2002). A nested PCR for detection of the repetitive fragment 529 bp (AF487550) of T. gondii was performed as previously described by Su and Dubey (2009). The Nc5 gen PCR was used for N. caninum PCR as described by Liddell et al. (1999) with minor modifications as described by Almería et al. (2002). The seroprevalence to T. gondii and N. caninum was estimated from the ratio of positive to the total number of samples, with binomial confidence intervals of 95%. Associations between the serological results and independent variables such as location and year of sample collection were analyzed using a Pearson’s chi-square test and by Fisher’s exact test when observations/category were <6. Differences between categories were analyzed using the Tukey test. Differences were considered statistically significant when P < 0.05. Statistical analyses were performed using SPSS 15.0 (Statistical Package for Social Sciences (SPSS) Inc.). Antibodies (MAT P 1:25) to T. gondii were found in 91 (80.5%; CI95%: 72.1–87.4) from 113 Common ravens sera with titers of 1:25 in 5 (5.5%), 1:50 in 22 (24.2%), 1:100 in 23 (25.3%), and P1:500 in 41 (45.1%). No statistically significant differences were found between prevalence of infection and age or location of trapped ravens. Statistically significant differences were observed among years of sampling, with the highest seroprevalence in the most recent years (2009–2010) compared to the 2005–2008 period (p = 0.007) (Table 1). N. caninum antibodies were detected by IFAT P 1:50 in 24 of the 67 (35.8%; IC95%: 24.5–48.5) sera analyzed. No statistically significant differences were found between prevalence of infection and age, location, and year of sampling (2009 and 2010) although a trend towards statistically significant differences was observed between years of sampling (p = 0.12) (Table 1). In the samples in which both pathogens were analyzed (n = 67), 20 samples were simultaneously positive to both protozoa, 42 were only positive to T. gondii and 4 were only positive to N. caninum. Only one sample was negative to both protozoa. A trend to-
Table 1 Seroprevalence of Toxoplasma gondii (MAT P 1:25) and Neospora caninum (IFAT P 1:50) in Common ravens (Corvus corax) from Catalonia (NE Spain). Factor
Total Age Location
Year
Category
61 year >1 year Centelles S.M. Centelles Oris Rupit i Pruit 2005 2006 2008 2009 2010
T. gondii (MAT)
N. caninum (IFAT)
Examined (n)
Positive (n)
Prevalence (%)
Examined (n)
Positive (n)
Prevalence (%)
113 67a 4 28 66 2 17 15c 15 14 49 18
91 60 3 22 53 2 12 8 10 9 46 16
80.5 89.5 75.0 78.6 80.3 100.0 70.6 53.3 66.7 64.3 93.9 88.9
67 59b 2 19 38 1 9 ND ND ND 49 18
24 23 0 9 13 0 2
35.8 38.9 0.0 47.4 34.2 0 22.2
15 9
30.6 50.0
MAT: modified agglutination test; IFAT: indirect fluorescent antibody test. a No age data from 42 samples. b No age data from 6 samples. c No data of year of sampling from 2 samples.
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wards a negative association between T. gondii and N. caninum was observed (Fisher exact test, P = 0.052). The presence of T. gondii and N. caninum DNA was not detected in the brain of the only two dead C. corax analyzed by PCR. To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in Common ravens in Spain and in the world. In addition, the prevalence of T. gondii antibodies found in Common ravens in Spain, in a relatively high number of analyzed samples (80.5% of 113 samples), is also one of the highest reported in wild birds worldwide (Dubey, 2002, 2009). Recent studies of T. gondii seroprevalence have shown a high prevalence of T. gondii antibodies in wild birds in Europe but did not examine Common ravens. Aubert et al. (2008) reported prevalence levels in raptors from France, that similar to our study were brought to rehabilitation centers, with levels of 79% of 14 Common buzzards (Buteo buteo), 50% of 12 Tawny owls (Strix aluco) and 11% of 18 Barn owls (Tyto alba). Lopes et al. (2011) reported a prevalence of antibodies in 50% of 52 wild birds brought to rehabilitation centers that include multiple species such as Black kite (Milvus migrans), Booted eagle (Hieraaetus pennatus), Common buzzard, Eurasian sparrowhawk (Accipiter nisus), Northern goshawk (Accipiter gentilis), Eurasian eagle owl (Bubo bubo) and Tawny owl in Portugal. The present results are also the first report of N. caninum antibodies in Common ravens from any country. The presence of birds on dairy farms with neosporosis abortion problems has been considered as a risk factor for N. caninum infection in some studies (Ould-Amrouche et al., 1999; Bartels et al., 1999), but information regarding involvement of wild birds in the epidemiology of N. caninum is scarce, especially in field conditions. The present study showed moderate to high seroprevalence levels of N. caninum antibodies in this species (35.8% of 67 C. corax). The fact that the analyzed ravens in the present study were captured in relation to attacks to farms, could have influenced the high seroprevalence levels observed, but is still a clear indication of the potential role of Common ravens as sentinels for both protozoan infections. A trend towards a negative association between T. gondii and N. caninum was observed. Possible explanations for this result may involve different exposure (with cattle farms being more probable sources of N. caninum, and sheep farms infected with T. gondii) or differences on immunity. Further studies are necessary to confirm this association. An interesting observation was that the highest seroprevalence levels in both T. gondii and N. caninum were observed in the most recent years of study. This could be an indication of an expansion of these protozoan infections in the affected farms over time, although other factors, such as differences in environmental conditions each year, could have also influenced these results. In addition, the prevalence levels observed in the present study corresponded mostly to juvenile Common ravens, since older ravens are difficult to trap. Another explanation could be that in this species, juveniles have a distinct foraging behavior from adults, and tend to group on feeding resources (Dall and Wright, 2009). This may explain high seropositivity levels in the juveniles of this study. Many studies in several species have shown that seroprevalence of T. gondii infection is age-related with higher seroprevalence in older animals (Dubey, 2009), probably due to a greater probability of exposure over time and long persistence of antibodies. Therefore, higher seroprevalence levels might be expected in older Common ravens in the same areas. The present results could indicate a role for Common ravens in the epidemiology of toxoplasmosis and neosporosis. However, detection and/or isolation of the parasite in tissues would be necessary to corroborate that Common ravens are an intermediate host of T. gondii and N. caninum. Unfortunately only two animals could be examined by PCR and both were negative. Further studies
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