Corvus Corax) in the Northeast of Spain

Research in Veterinary Science 93 (2012) 300–302

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Research in Veterinary Science

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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 b Servei d’Ecopatologia de Fauna Salvatge (SEFaS), Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain c 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 article info abstract

Article history: In recent years, multiple cases of aggressive behavior of Common ravens (Corvus corax) have been Received 29 April 2011 reported by farmers in Catalonia (NE Spain), including attacking of newborn animals and consumption Accepted 12 May 2011 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 Keywords: were found in 24 (35.8%; IC 95%: 24.5–48.5) of 67 Common ravens tested by an indirect fluorescence anti- Common ravens body test with titers ranging from 1:50 (n = 18) to P1:100 (n = 6). To the author’s knowledge, this is the Seroprevalence first report of the presence of T. gondii and N. caninum antibodies in C. corax. The seroprevalence detected Neospora caninum Toxoplasma gondii 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 ing birds, and is zoonotic (Dubey, 2009). Felids are the only defin- Northern raven, is a large, all-black passerine bird in the crow fam- itive hosts of T. gondii and they excrete environmentally resistant ily. Found across the northern hemisphere, it is the most widely oocysts. Cats are thought to become infected with T. gondii via pre- distributed of all corvids. Common ravens are extremely versatile dation on infected birds and rodents. T. gondii infections are preva- and opportunistic in finding sources of nutrition. They are mainly lent in many avian species, and it can cause mortality in some scavengers feeding on carrion, insects, cereal grains, berries, fruit, species of birds (Dubey, 2002), including the endangered ‘Alala or small animals, and food waste. They are also known to eat the Hawaiian crow (Corvus hawaiiensis)(Work et al., 2000). Viable T. afterbirth of ewes and other large mammals (Boarman and Hein- gondii has been isolated from the American crow (Corvus brachy- rich, 1999). The Common raven has coexisted with humans for rhynchus) from the USA (Finlay and Manwell, 1956), Carrion crow thousands of years and in some areas is so numerous that it is con- (Corvus corone) from Kazakhstan (Pak, 1976) and Slovakia (Catár, sidered a pest. Recently, multiple cases of aggressive behavior of 1974), Jackdaw (Corvus monedula) and Rook (Corvus frugilegus) Common ravens have been reported by farmers in Catalonia (NE from Czech Republic (Uterák et al., 1992). It is noteworthy, that Spain), including attacking of newborn animals and consumption these studies were conducted several decades ago. of dead foetuses in farms. N. caninum is considered one of the most important causes of Toxoplasma gondii and Neospora caninum are two closely-re- abortion in cattle worldwide (Dubey et al., 2007). Although ca- lated, intra-cellular apicomplexans of worldwide distribution that nids (dogs, coyotes, dingoes) are the definitive hosts for N. cani- have been implicated in abortion and reproductive disorders in num many aspects of transmission of this parasite remain livestock (Dubey et al., 2007; Dubey, 2010). Toxoplasmosis, caused unknown. The presence of birds in cattle raising farms has been by T. gondii, affects most species of warm-blooded animals, includ- associated with outbreaks of abortion and considered as a risk factor for N. caninum infection (Ould-Amrouche et al., 1999; Bar- tels et al., 1999). Recently the domestic chicken (Gallus domesti- ⇑ Corresponding author at: Departament de Sanitat i Anatomia Animals, Facultat cus) and the sparrow (Passer domesticus) were reported as de Medicina Veterinaria, Universitat Autònoma de Barcelona, 08193 Bellatera, natural intermediate hosts for N. caninum (Costa et al., 2008; Barcelona, Spain. E-mail address: [email protected] (S. Almería). Gondim et al., 2010).

Investigation of T. gondii and N. caninum exposure in birds could IgY antibodies exhibit good cross-reactivity with most avian spe- be a useful way to assess environmental contamination with oo- cies (Cray and Villar, 2008) and has been validated for the detec- cysts, since some avian populations feed directly on the ground tion of serum immunoglobulins in wild birds of different species and are continuously exposed to oocyst ingestion (Gondim et al., by ELISA (Martínez et al., 2003). A cut-off of 1:50 or higher was 2010). In addition, investigation of T. gondii and N. caninum infec- considered positive, as previously described for domestic and wild- tion in scavenger wild birds could also assess the possibility of con- life species (Ortuño et al., 2002; Almería et al., 2007). Only animals tact with intermediate hosts and the associated risk for public trapped in 2009 and 2010 (n = 67) were available for N. caninum health. antibody detection. We report for the first time T. gondii and N. caninum infections DNA was extracted from approximately 1 g of brain tissue from in the C. corax. the two dead Common ravens. DNA was extracted as previously A total of 115 wild Common ravens were trapped in Catalonia, described by Almería et al. (2002). A nested PCR for detection of NE Spain and brought to the rehabilitation center ‘‘Centro de Recu- the repetitive fragment 529 bp (AF487550) of T. gondii was per- peración de Fauna Salvaje (CRFS) of Torreferrussa (Santa Perpetua formed as previously described by Su and Dubey (2009). The Nc5 de Moguda, Barcelona, Spain) between 2006 and 2010. The birds gen PCR was used for N. caninum PCR as described by Liddell were captured with government license after farmers complained et al. (1999) with minor modifications as described by Almería of attacks to their animals, in particular neonates and dead foetus- et al. (2002). es. The species is protected by Spanish national laws and therefore The seroprevalence to T. gondii and N. caninum was estimated only blood samples were collected by venipuncture of right jugular from the ratio of positive to the total number of samples, with vein from trapped ravens, in compliance with the Ethical Principles binomial confidence intervals of 95%. Associations between the in Animal Research of the CRFS of Torreferrussa. All crows were serological results and independent variables such as location apparently healthy and were released after sampling. Only two and year of sample collection were analyzed using a Pearson’s Common ravens died after trapping and brain tissues were col- chi-square test and by Fisher’s exact test when observations/cate- lected from the dead birds for detection of the parasite DNA. Sera gory were <6. Differences between categories were analyzed using from these two animals were not available for analysis. Data were the Tukey test. Differences were considered statistically significant collected regarding location and year of sampling. Age was deter- when P < 0.05. Statistical analyses were performed using SPSS 15.0 mined whenever possible by external characters, with 94.4% of (Statistical Package for Social Sciences (SPSS) Inc.). the birds (67 of 71 birds with age available) being juveniles (61 Antibodies (MAT P 1:25) to T. gondii were found in 91 (80.5%; calendar year). Blood was placed in a serum collection tube until CI95%: 72.1–87.4) from 113 Common ravens sera with titers of clotted and then centrifuged. Sera were stored at À20 °C until 1:25 in 5 (5.5%), 1:50 in 22 (24.2%), 1:100 in 23 (25.3%), and analyzed. P1:500 in 41 (45.1%). No statistically significant differences were Antibodies to T. gondii were determined by the modified agglu- found between prevalence of infection and age or location of tination test (MAT) using 2-mercaptoethanol and formalin-fixed trapped ravens. Statistically significant differences were observed whole tachyzoites as described previously (Dubey and Desmonts, among years of sampling, with the highest seroprevalence in the 1987). Sera were tested at 1:25, 1:50, 1:100 and 1:500 dilutions. most recent years (2009–2010) compared to the 2005–2008 period A commercial positive control (Toxotrol-A, Biomerieux, France) di- (p = 0.007) (Table 1). luted from 1:25 to 1:3200 (with a minimum titer of 1:200) as well N. caninum antibodies were detected by IFAT P 1:50 in 24 of as negative controls were included in each test. Titers of 1:25 or the 67 (35.8%; IC95%: 24.5–48.5) sera analyzed. No statistically sig- higher were considered positive and those with doubtful results nificant differences were found between prevalence of infection were re-examined. This technique has been successfully tested in and age, location, and year of sampling (2009 and 2010) although several bird species (Dubey, 2002). a trend towards statistically significant differences was observed Antibodies against N. caninum were assayed using an indirect between years of sampling (p = 0.12) (Table 1). fluorescent antibody test (IFAT) using slides coated with whole N. In the samples in which both pathogens were analyzed (n = 67), caninum tachyzoites (VMRD, Pullman, Washington, USA), and FITC 20 samples were simultaneously positive to both protozoa, 42 labeled rabbit anti-chicken IgY (IgG) (whole molecule) (SIGMA, were only positive to T. gondii and 4 were only positive to N. cani- Spain) at 1:200 dilutions. This commercially available anti-chicken num. 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 Category T. gondii (MAT) N. caninum (IFAT) Examined (n) Positive (n) Prevalence (%) Examined (n) Positive (n) Prevalence (%) Total 113 91 80.5 67 24 35.8 Age 61 year 67a 60 89.5 59b 23 38.9 >1 year 4 3 75.0 2 0 0.0 Location Centelles 28 22 78.6 19 9 47.4 S.M. Centelles 66 53 80.3 38 13 34.2 Oris 2 2 100.0 1 0 0 Rupit i Pruit 17 12 70.6 9 2 22.2 Year 2005 15c 8 53.3 ND 2006 15 10 66.7 ND 2008 14 9 64.3 ND 2009 49 46 93.9 49 15 30.6 2010 18 16 88.9 18 9 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. 302 R. Molina-López et al. / Research in Veterinary Science 93 (2012) 300–302 wards a negative association between T. gondii and N. caninum was should analyze the presence of T. gondii and N. caninum DNA in tis- observed (Fisher exact test, P = 0.052). sues and confirm that the Common ravens are intermediate host in The presence of T. gondii and N. caninum DNA was not detected the cycle of both parasites. in the brain of the only two dead C. corax analyzed by PCR. In conclusion, the high seroprevalence levels observed in the To our knowledge, this is the first report of T. gondii and N. cani- present study indicate a high exposure to both T. gondii and N. caninum seroprevalence in Common ravens in Spain and in the world. num in Common ravens from Catalonia, Northeast Spain, and sug- In addition, the prevalence of T. gondii antibodies found in Com- gest an important role for this species in the epidemiology of both mon ravens in Spain, in a relatively high number of analyzed sam- parasites. ples (80.5% of 113 samples), is also one of the highest reported in This study received partial support from the Spanish CICYT wild birds worldwide (Dubey, 2002, 2009). Recent studies of T. Grant AGL2007-65521 C02/GAN. The authors thank Dr. Pita Gon- gondii seroprevalence have shown a high prevalence of T. gondii dim for the generous donation of chicken N. caninum positive con- antibodies in wild birds in Europe but did not examine Common trol sera for IFAT. We thank all the staff of the Torreferrussa ravens. Aubert et al. (2008) reported prevalence levels in raptors Rehabilitation Center for their devoted care of the patients. from France, that similar to our study were brought to rehabilitation centers, with levels of 79% of 14 Common buzzards (Buteo bu- References teo), 50% of 12 Tawny owls (Strix aluco) and 11% of 18 Barn owls (Tyto alba). Lopes et al. (2011) reported a prevalence of antibodies Almería, S., Ferrer, D., Pabón, M., Castellà, J., Mañas, S., 2002. Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum. 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Bartels, C.J., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum on dairy farms with neosporosis abortion problems has been con- associated abortion storms in dairy herds in The Netherlands (1995 to 1997). sidered as a risk factor for N. caninum infection in some studies Theriogenology 52, 247–257. (Ould-Amrouche et al., 1999; Bartels et al., 1999), but information Boarman, W., Heinrich, B., 1999. Corvus corax: Common raven. The Birds of North America 476, 1–32. regarding involvement of wild birds in the epidemiology of N. cani- Catár, G., 1974. Toxoplazmóza vekologickych podmienkach na Slovensku. num is scarce, especially in field conditions. The present study Biologické Práce (Bratislava) 20, 1–138. showed moderate to high seroprevalence levels of N. caninum anti- 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. 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