Neospora caninum in crows from Israel

Veterinary Parasitology 212 (2015) 375–378

Contents lists available at ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Short communication

Neospora caninum in crows from Israel H. Salant a,∗ , M.L. Mazuz b , I. Savitsky b , A. Nasereddin c , E. Blinder b , G. Baneth a a

School of Veterinary Medicine, Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel Department of Parasitology, Kimron Veterinary Institute, PO Box 12, Bet Dagan 50250, Israel c Al-Quds Nutrition and Health Research Institute (ANAHRI), Medical School, Al-Quds University, East Jerusalem, Abu-Deis, PO Box 19356, Palestine b

a r t i c l e

i n f o

Article history: Received 8 June 2015 Received in revised form 16 August 2015 Accepted 18 August 2015 Keywords: Neospora caninum MAT IFAT PCR Crows Israel

a b s t r a c t A cross-sectional Neospora caninum seroprevalence study was performed on free ranging crows (Corvus cornix, Corvus monedula and Corvus splendens) from Israel in order to assess their exposure to this pathogen and evaluate their role as potential hosts or as sentinels of infection. Using the modified agglutination test (MAT) with a cutoff titer of 1:100, 30 out of 183 crows (16.4%) were found to be N. caninum seropositive. Positive results were validated and confirmed by the indirect fluorescent antibody test (IFAT). There was 100% agreement between tests when cut-off titers of 1:50 and 1:100 were applied for the IFAT and MAT, respectively. PCR analysis of brain extracts from all crows resulted in the detection of N. caninum DNA for the first time in crows belonging to two species, C. cornix and C. monedula. The high N. caninum seroprevalence in crows suggests that widespread exposure to infection with N. caninum exists especially in central and northern Israel and that crows may act as suitable markers for disease prevalence in the areas in which they are found. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Neospora caninum is an apicomplexan parasite of worldwide distribution that infects a wide range of animals, including cattle, dogs, sheep, goats, horses, different species of wild animals and birds (Donahoe et al., 2015). This protozoan parasite is considered to be an important cause of bovine abortions, stillbirths and reproductive failure in cattle worldwide (Dubey, 2003). Cattle seropositive to N. caninum have been found to be 3–11 times more likely to abort than seronegative animals (Almeria and López-Gatius, 2013). More than 45% of dams in Israeli dairy farms have been observed to carry specific N. caninum antibodies and 18% of abortions were associated with N. caninum infection, as confirmed by serological and molecular assays on aborted fetuses (Fish et al., 2007; Mazuz et al., 2011). Although N. caninum may be transmitted horizontally to cattle after ingesting infective oocysts shed by the definitive hosts, dogs and wild canids (McAllister et al., 1998), transplacental infection from persistently infected dams is known to be the main route of infection in dairy cattle (Dubey, 2003). Bartels et al. (2007) have reported that the presence of dogs on dairy farms significantly increased the probability of cattle to be

∗ Corresponding author. Fax: +972 2 6757425. E-mail addresses: [email protected], [email protected] (H. Salant). http://dx.doi.org/10.1016/j.vetpar.2015.08.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.

infected by horizontal transmission. It is therefore crucial to determine the level of environmentally dispersed oocysts in order to evaluate the risk of this life cycle stage as a transmission mode for animal exposure to N. caninum. Crows of several species are commonly found in all inhabited parts of the world and are abundant in many regions. They are extremely successful in their ability to establish large populations in urban and rural areas and feed on almost any source of nutrition including carrion, insects, grains, berries, fruit, small birds and other animals. Crows tend to remain in the same territory persistently. Since crows are scavengers, carnivores and ground feeders, they may become infected by oocyst contaminated food, or also ingest tissues containing N. caninum cysts. Infected birds would serve as good sentinels for markers of N. caninum presence and contamination of the environment with this parasite. A study by Molina-López et al. (2012) in the north–east of Spain reported that antibodies to N. caninum were found in 24 (35.8%; IC 95%: 24.5–48.5) of 67 common ravens (Corvus corax) tested by an indirect fluorescence antibody test with baseline titers from 1:50. DNA extracted from two crow brains and examined for N. caninum were both found to be negative in the same study. The objective of this study was to estimate the seroprevalence of N. caninum in Israeli crows and to evaluate crow tissue infection by PCR.

376

H. Salant et al. / Veterinary Parasitology 212 (2015) 375–378

2. Materials and methods

wildlife species (Almería et al., 2007). Sera were diluted two fold to a final titer of 1:12800.

2.1. Birds 2.4. PCR One hundred and eighty three crows of the species Corvus cornix (Hooded crow; n = 162), Corvus monedula (Jackdaw; n = 5) and Corvus splendens (House crow; n = 16; Table 1) were included in the survey. Crows sampled were all juvenile birds that had hatched approximately 2–4 months previously but were able to fly and feed independently. Crows were captured using Australian Traps and sacrificed humanely by means of intracardiac pentobarbital injection (200 mg/ml; Pental, CTS, Israel), as permitted by the Israeli Nature and Parks Authority, during January to November 2011 and August to October 2013. Sera and brain specimens were collected from each individual crow and stored at −20 ◦ C. In addition, spleen, liver, lung, heart, and pectoral muscle samples were collected from sixty two of the 183 crows. 2.2. Sampling locations Crows were captured in areas that ranged from Haifa, (32◦ 49 49.30 N) in northern Israel, to Eilat, (29◦ 33 27.60 N) in the south. Crow sampling regions were divided into 3 geographical regions; (1) Haifa (32◦ 49 49.30 N) in the north; (2) A central region comprised of the area between Beeri (31◦ 40 0 N) to Gaash (32◦ 13 40 N) and, (3) Eilat (29◦ 33 27.60 N) in the south.

2.4.1. DNA extraction One hundred and fifty milligrams of tissue material from crows was added to 200 ␮l of lysis buffer (50 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, and 200 ␮g/ml of proteinase K) and incubated at 60 ◦ C for 2 h. Nucleic acids were extracted twice using phenol followed by chloroform/isoamyl alcohol. DNA was eluted in 100 ul of TE (Tris-EDTA pH 8.0) buffer (Ausubel et al., 1994). 2.4.2. Nested PCR (nPCR) amplification Detection of N. caninum DNA was performed by nPCR as previously described by Fish et al. (2007), using two sets of primers, located on gene, Nc5, and based on the DNA sequence of N. caninum GenBank accession number, X84238. The PCR reaction contained 1 ␮l of genomic DNA, each primer concentration 0.4uM and 1× ReddyMix (Thermo Fisher Scientific Inc., UK) in a final volume of 25 ␮l. Samples from N. caninum isolate, NcIs491 and negative (no DNA) were added to each reaction as positive and negative controls, respectively. Products were visualized on 1.5% ethidium bromide stained agarose gels. Positive PCR products (299 bp) were purified with the PCR purification kit (Qiagen, CA, USA), sequenced, and submitted to the GenBank.

2.3. Serological examination 3. Results 2.3.1. The modified agglutination test (MAT) MAT for N. caninum antibodies was carried out as previously described (Packham et al., 1998). N. caninum tachyzoites were prepared from the Israeli isolate NcIS491 (Fish et al., 2007) collected from media of heavily infected Vero cells. Two positive bovine sera samples and two negative controls (one negative bovine serum sample and one PBS without serum), were included in each plate. Incubation at 37 ◦ C was performed overnight. Sera from birds were diluted serially two fold in PBS from 1:25 to a final dilution of 1:12800. Titers of 1:100 or higher were considered positive. 2.3.2. Indirect fluorescent antibody test (IFAT) All MAT positive sera samples (titers ≥ 1:100) and four negative crow serum samples were confirmed by performing IFAT. Presence of antibodies to N. caninum by IFAT was tested according to Fish et al. (2007) with the exception that FITC labeled rabbit anti-chicken IgY (IgG; whole molecule; Sigma, Israel) was used as a secondary antibody at a concentration of 1:200. A cut-off titer of 1:50 or higher was considered positive, as previously described for domestic and

Thirty out of 183 (16.4%) crow sera were seropositive for N. caninum (Table 1). The N. caninum seroprevalence among crows in the north, center and south of Israel was 16.1%, 19.1% and 0%, respectively (Table 1). All MAT positive (titers ≥ 1:100) and four MAT negative defined sera were confirmed using the IFAT. Notably, one crow from Kibbutz Beeri was highly seropositive with a titer of 1:12800. Two crows from locations in central Israel, Kibbutz Beeri (C. cornix; denoted GenBank accession number KR858303) and Ganei Yavne (C. monedula; denoted GenBank accession number KR858302), were found positive by PCR from brain tissue. The two hundred and ninety nine bp sequences from each crow were deposited in GenBank. The DNA sequences analyzed using the BLAST utility (http://www.ncbi.nlm.nih.gov/BLAST) displayed 99% identity with N. caninum (GenBank accession numbers KF649846.1, and EF463098.1, respectively). Both of the PCR positive crows were seronegative by the MAT and IFAT assays. None of different organ tissue samples collected from sixty two crows was positive for N. caninum by PCR.

Table 1 Serological results of N. caninum in crows according to sampling site and region in Israel using the modified agglutination test (MAT) with comparison to the indirect fluorescent antibody test (IFAT). Sampling point (crow species)

Carmel forest (Corvus cornix) Haogen (C. cornix) Gaash (C. cornix) Raanana (C. cornix) Ganei Yavne (C. monedula) Kibbutz Beeri (C. cornix) Jerusalem (C. cornix) Eilat (C. splendens) Total a

Final MAT and IFAT (in brackets) titers

1:100 (1:50)

1:200 (1:100)

1:400 (1:200)

1:800 (1:400)

0 0 0 0 0 1(1) 0 0 1(1)

6 (6) 3 (2) 0 7 (6) 0 0 0 0 20 (14)

0 0 (1) 4 (2) 2 (3) 0 0 0 0 6 (6)

1(1) 0 4 (6) 0 1(1) 1a (1) 0 0 3 (9)

This serum sample was still reactive at the highest MAT limit of 1:12800.

Positive PCR

Seroprevalence rate (%)

Region in Israel Rate (%)

0 0 0 0 1/5 1/21 0 0 2/183

7/52 (13.5) 3/10 (30) 8/14 (57) 9/40 (22.5) 1/5 (20) 2/21 (9.5) 0/25 (0) 0/16 (0) 30/183 (16.4)

North 10/62 (16.1) Center 20/105 (19.1)

South 0/16 (0) 30/183 (16.4)


4. Discussion Serological analyses of 183 crows showed that seropositivity to N. caninum was found in 30 out of 183 (16.4%) crows. The seropositive crows were from central and northern Israel but not from the southern part of the country. The lower seroprevalence of N. caninum infection in Israeli crows compared to those sampled in Spain (24/67; 35.8%, Molina-López et al., 2012) may be attributed to differences in their exposure to environmentally dispersed oocysts from canids in the regions that are in close proximity to affected dairy herds, environmental conditions that are related to precipitation and maximum daily temperatures, or differences in age or resistance to infection between the various crow species sampled. Regional differences in N. caninum seroprevalence were noticed. In the central and northern regions of Israel high seroprevalence (19.1% and 16.1%, respectively) were observed. However in Eilat, no seropositive crows were found (0/16). Eilat is located in an arid desert area. Therefore, it is possible that the climate of Eilat may not be suitable for sporulated oocysts to remain viable or for infected tissue to remain infective for long periods of time so as to infect birds further. Alves Neto et al. (2011) have shown that the environmentally resistant N. caninum oocysts can only be destroyed by high temperatures of 100 ◦ C for 1 min similar to Toxoplasma gondii. For example, infectivity of T. gondii oocysts at temperatures of 10–25 ◦ C did not deteriorate for at least 200 days, but at 40 ◦ C they were infective for only 9 days and at 45 ◦ C only for 1 day (Dubey, 1998). Also, the dog population density in this drier region of Eilat is lower (number of registered dogs in 2014 was 4950) than other central urbanized regions of this country (31,700 in Tel Aviv; 12,300 in Haifa in 2014; http://www.vetserv.moag.gov.il/Vet/shirutim/ rishum klavim vehisun kalevet/iun klavim.htm), therefore reducing the number of potentially infected definitive hosts to shed oocysts. In addition, the lower N. caninum seroprevalence in Eilat may have been due to possible resistance of the crow species found In Eilat, C. splendens to infection by this parasite or due to a lower number and density of dairy cattle in this area. The MAT assay was used for serological testing because of its ease in preparation and the simplicity of analyzing test results. The commercially available anti-chicken IgY antibody exhibits 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 the enzyme linked immunosorbent assay (ELISA) (Martínez et al., 2003). In addition, Packham et al. (1998) have found high sensitivity and specificity of results when compared to ELISA and IFAT methods. Although the baseline titer of 1:100 for MAT would intuitively appear high for this assay, it was used to reduce the possibility of false positive results and was confirmed in 100% of all tested samples with the IFAT in our study. We are not aware of any previous publication that has reported the detection of N. caninum in crow species, C. cornix (GenBank accession KR858303) and C. monedula (GenBank accession KR858302). Two other studies have also reported finding PCR positive crows from the genus Corvus. Darwich et al. (2012) observed that 2 of 33 (6%) magpie brain samples (Pica pica) in Spain were N. caninum positive, and Muz et al. (2014) found 2 of 24 (8%) sampled carrion crows (Corvus corona) in Turkey positive. Neither of these two studies performed simultaneous serological studies for the same birds. The finding of two serologically negative crows with positive PCR brain tissues is an interesting finding. False seronegative samples were ruled out by repeating the serological samples using another assay, the IFAT. The reason that PCR positive crows in this study were not simultaneously serologically positive could be explained by a number of possible reasons. Firstly, the PCR positive crows may have been sampled in the window period before

377

they seroconverted. Another explanation could be that these birds were juveniles and refractory to the production of detectable antibody responses against N. caninum. Lastly, it has previously been shown that after N. caninum experimental infection of pigeons and chickens, an abrupt seroconversion to the parasite occurred. However, after a short period of seropositivity there was a drop in the antibodies to an undetectable level (Mineo et al., 2009). A general limitation of seroprevalence studies in wildlife, including the molecular demonstration of N. caninum in individual animals is that serological and PCR positivity are not necessarily analogous to the presence of viable organism in the host (Donahoe et al. 2015). Other tests such as culture or bioassay would better confirm positivity of infected animals. In conclusion, exposure and molecular detection of crows with N. caninum in Israel has been demonstrated. The successful detection of parasite DNA by PCR and its further sequencing was possible for crows of two species, C. cornix and C. monedula. Since dispersed infective oocysts and infected carrion, stillbirth or aborted fetuses or placentas in contaminated environments are likely to be responsible for widespread infection of these scavenger birds, crows may be potentially effective sentinels for N. caninum presence in the vicinity of dairy and beef farms. Their contribution to the N. caninum life cycle needs to be investigated further especially due to the fact that they may further transmit infection to other carnivorous definitive hosts when consumed post-mortally.

Acknowledgements The authors would like to thank the Israel Parks Authority veterinarian, Dr. Roni King, and his team of field inspectors for their cooperation and contribution of trapped birds for sampling.

References 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. Vet. Parasitol. 143, 21–28. Almeria, S., López-Gatius, F., 2013. Bovine neosporosis: clinical and practical aspects. Res. Vet. Sci. 95 (2), 303–309. Alves Neto, A.F., Bandini,L, A., Nishi,S, M., Soares, R.M., Driemeier, D., Antoniassi, N.A.B., Schares, G., Gennari,S, M., 2011. Viability of sporulated oocysts of Neospora caninum after exposure to different physical and chemical treatments. J. Parasitol. 97, 135–139. Ausubel, R., Kingston, R.E., Moore, D.D., Scidman, J.G., Smith, K. (Eds.), 1994. Current Protocols in Molecular Biology, Nov 14. John Wiley & Sons. Bartels, C.J.M., Huinink, I., Beiboer, M.L., van Schaik, G., Wouda, W., Dijkstra, T., Stegeman, A., 2007. Quantification of vertical and horizontal transmission of Neospora caninum infection in Dutch dairy herds. Vet. Parasitol. 148, 83–92. Cray, C., Villar, D., 2008. Cross-reactivity of anti-chicken IgY antibody with immunoglobulins of exotic avian species. Vet. Clin. Pathol. 37, 328–331. Darwich, L., Cabezón, O., Echeverria, I., Pabón, M., Marco, I., Molina-Lopez,R, Alarcia-Alejos, O., Lopez-Gatius,F, Lavín, S., Almeria,S, 2012. Presence of Toxoplasma gondii and Neospora caninum DNA in the brain of wild birds. Vet. Parasitol. 183, 377–381. ˇ Donahoe, S.L., Lindsay, S.A., Krockenberger, M., Phalen, D., Slapeta, J., 2015. A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. Int. J. Parasitol Parasites Wildl. 4, 216–238. Dubey, J.P., 1998. Toxoplasma gondii oocyst survival under defined temperatures. J. Parasitol. 84, 862–865. Dubey, J.P., 2003. Review of Neospora caninum and neosporosis in animals. Korean J. Parasitol. 41, 1–16. Fish, L., Mazuz, L.M., Molad, T., Savitsky, I., Shkap, V., 2007. Isolation of Neospora caninum from dairy zero grazing cattle in Israel. Vet. Parasitol. 149, 167–171. 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. Funct. Ecol. 17, 700–706. Mazuz, L.M., Fish, L., Molad, T., Savitsky, I., Wolkomirsky, R., Leibovitz, B., Shkap, V., 2011. Neospora caninum as causative pathogen of abortion in cattle. Isr. J. Vet. Med. 66, 14–18. 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.

378


Mineo, T.W., Carrasco, A.O., 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. Molina-López, R., Cabezón, O., Pabón, M., Darwich, L., Obón, E., Lopez-Gatius, F., Dubey, J.P., Almería, S., 2012. High seroprevalence of Toxoplasma gondii and Neospora caninum in the Common raven (Corvus corax) in the Northeast of Spain. Res. Vet. Sci. 93, 300–302.

Muz, M.N., Orunç Kilinç, O., I˙ s¸ler, C.T., Altu˘g, E., Karakavuk, M., 2014. Molecular diagnosis of Toxoplasma gondii and Neospora caninum in brain tissues of some wild birds. Fakultesi Dergisi. 21, 173–178. Packham, A.E., Swerlow, K.W., Conrad, P.A., Loomis, E.F., Rowe, J.D., Anderson, M.L., Marsh, A.E., Cray, C., Barr, B.C., 1998. A modified agglutination test for Neospora caninum: development, optimization, and comparison to the indirect fluorescent-antibody test and enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol., 467–473.