ABSTRACT

Objective:

Neospora caninum is a protozoon parasite that has a worldwide distribution and mainly causes abortion in cattle and current serological evidence shows that the disease may be also zoonotic. Wild birds play a role as a reservoir of the disease in nature. The study aimed to determine the prensence of N. caninum in wild birds.

Methods:

In this study, the presence of neosporosis in wild birds (n=55) including 22 different species found in the western side of Turkey, was investigated by polymerase chain reaction (PCR). In addition, PCR positive samples were confirmed by sequencing, BLAST, and phylogenetic analysis using MEGA7.

Results:

Obtained results showed that the presence of N. caninum DNA was 5.45% (3/55) in brain-heart homogenates wild birds. The bird species which were found positive for N. caninum were little owl (Athene noctua), common buzzard (Buteo buteo), and little tern (Sternula albifrons). According to phylogenetic analysis and BLAST, all samples were compatible with reference N. caninum isolates.

Conclusion:

To the best of authors’ knowledge, this is the first study detecting N. caninum in little tern. In future studies, it may be interesting to investigate the prevalence of N. caninum in other wild animals to elucidate the transmission properties.

INTRODUCTION

Neospora caninum (N. caninum) is a protozoon parasite that can cause neurological disorders, paralysis, and skeletal muscle problems in dogs. Cattle are by far the most affected species by this parasite because of abortions (1-3).

Up to date, viable parasites have been isolated only from dogs, gray wolves, cattle, sheep, water buffalo, europian bison, and white-tailed deer. However, many studies using molecular techniques showed that N. caninum can be detected in other mammals and birds (4-11). In humans, although antibodies against this parasite have been described, the zoonotic potential is unclear because neither parasite nor its DNA has not been detected in human tissues (4,12).

N. caninum was first described by Bjerkas and Presthus (13) from dogs in Norway in 1988 and later in a wide range of other animals (14). In wild bird species, N. caninum infection is transmitted by hunting and eating small animals containing tissue cyst or their carcasses, as well as through consuming food and water contaminated by oocyst. Thus, predator birds are more frequently infected by N. caninum. Different researches have highlighted the role of birds in the transmission of N. caninum (1,15,16). In experimental animal models using chickens, partridges, and quails, N. caninum caused death in all of the partridges and neurological disorders as well as dose-dependent variable mortality rates in chickens and quails (17-19).

Wild birds require different areas for hunting, feeding, and breeding and these properties are very important for the transmission of N. caninum in nature (20-22). Turkey has important geographical locations on the migration routes of wild birds and also hosts them. İzmir and Manisa provinces are located in the western side of Turkey next to the migration routes of wild birds. Moreover, İzmir is the third biggest city in Turkey and has a huge wildlife park and bird sanctuary. The present study aimed to investigate the presence of N. caninum DNA in deceased wild birds found in the western part of Turkey by polymerase chain reaction (PCR).

METHODS

Characteristics of the Study Area

İzmir and Manisa provinces (latitude: 37-38 °N, longitude: 26-29 °E) which have a mild Mediterranean climate and are located on the western part of Turkey which have a neighborhood to the Gediz Delta Plain. İzmir Bird Paradise sheltering 289 species of birds has a size of 8.000 hectares. Overall, 50 thousand birds come to this region during migrating. In addition, İzmir bird paradise is the popular resting and hunting area of wild birds coming to Gediz Delta Plain.

Birds and Sample Collection

Dead wild birds were found in the İzmir Bird Paradise or brought to İzmir Natural Life Park Clinics from eight different districts of İzmir province (Bornova, Konak, Bergama, Kemalpaşa, Balçova, Karabağlar and Seferihisar) and four districts from Manisa province (Saruhanlı, Turgutlu, Salihli and Centrum) between 2015 and 2017. İzmir Bird Paradise and İzmir Natural Life Park are located in Çiğli district next to Gediz Delta Plain. Organs (brain and hearth) were collected from 55 dead birds. The species of wild birds are listed in Table 1.

Organ Homogenization and DNA Extraction

The organs homogenates were prepared both the brain and heart of wild birds as previously described (23-25). Firstly, the brain and heart of the dead birds were weighed together. 125 mL (125 mL NaCl/10 gr organs) 0.9% NaCl was added and homogenized using a blender (Waring, USA). Thereafter, trypsin was added to the homogenate (0.5 gr trypsin/10 gr organs) and incubated with 120 rpm at 37 °C for 60 min in an incubator shaker (New Brunswick, USA). After incubation, the homogenate was filtered through sterile two-layered gauzes and centrifuged at 910×g. Next, the supernatant was discarded and the pellet was washed two times with 0.9% NaCl. After the last centrifugation, the pellet was homogenized with 5 mL 0.9% NaCl and, 500 µL aliquot was kept for DNA extraction. DNA isolation from bird organ samples was performed using QIAamp DNA Mini Kit (Qiagen, USA) according to the manufacturer’s protocol.

Molecular Detection

PCR targeting a ~340 bp product of the N. caninum Nc5 gene was performed as described using the Np6 (Forward primer, 5’-CTCGCCAGTCAACCTACGTCTTCT-3’) and Np21 (Reverse primer, 5’-CCCAGTGCGTCCAATCCTGTAAC-3’) primers (1,26). The 20 µL amplification reactions included 2 µL template DNA, the primers (0.5 µM each), 1.25 U Taq DNA Polymerase (Thermo Scientific, USA), 200 µM dNTPs, 3.25 mM MgCl₂ and 1xTaq reaction buffer. The PCR amplification reaction was performed using the following protocol: 10 min initial denaturation step at 95 ^oC, followed by 40 cycles of 60 s at 95 ^oC, 60 s at 56 ^oC, and 60 s at 72 ^oC, and a final extension of 10 min at 72 ^oC. All PCR products were separated by 1.5% agarose gel electrophoresis, stained by ethidium bromide and, visualized under DNR bio-imaging systems (Israel). A DNA sample confirmed to be belonging to N. caninum was used as the positive control (1) and, distilled water was used as the negative control.

Sequencing

PCR products of 340 bp fragments belonging to positive samples were sequenced by ABI3730XL. Generated sequences were edited and aligned by MEGA 7.0 software. Also, BLAST analysis was performed to compare with reference N. caninum samples in National Center for Biotechnology Information (NCBI).

The phylogenetic analysis was performed by MEGA 7.0 software. The phylogenetic tree based on Nc5 gene sequences belonging to Neospora isolates was constructed by MEGA7.0 software according to the Neighbour Joining/Maximum Likelihood method using Kimura 2 Gamma distribution (K2+G) model with 500 Bootstrap replications.

RESULTS

According to the PCR results, N. caninum Nc5 gene was detected in organ samples of three birds (3/55, 5.45%) (Figure 1) among all wild birds. The presence of N. caninum was 2.5% (2/40) among predator wild birds. In the remaining birds, presence of N. caninum was 16.6% (1/15). This birds are not predators but eat fish, insects, and reptiles. N. caninum positive bird species were a little owl, common buzzard, and little tern.

Among the N. caninum positive birds, the location of common buzzard was unknown, others were detected in the Çiğli (İzmir Bird Sanctuary) district (Figure 2).

Nc5 amplicons from positive samples were sequenced using the Np5-Np21 primer pair, and N. caninum positivity was confirmed. The Nc5 genomic DNA region displayed homology at levels of 96%, 97%, and 98%, respectively, with a N. caninum isolated from naturally infected cattle from Italy (KP715563.1).

Phylogenetic analysis results were compatible with BLAST and N. caninum isolates were more close to KP715563.1 (Cattle 2) than other N. caninum isolates (Figure 3). Similarity rates changed from 96% to 98.07%.

DISCUSSION

N. caninum leads to major economic losses in livestock worldwide. For example, N. caninum causes 2.38 billion dollars’ losses per year in the USA due to abortions in cattle (15). In addition, it was reported in Spain that abortions rates in sheep and goats caused by N. caninum were 6.75% and 12.5% (27).

Globally, the prevalence of N. caninum varies between 1.5-83.6% in wild birds using PCR or serological assays in distinctive regions of the world (4). In Brazil, N. caninum DNA was detected in six out of 100 free-range chickens (28). In a study conducted in Iran, the prevalence of N. caninum using PCR was 8% in sparrows (29). In Spain, N. caninum DNA was detected in three birds (1.5%; n=200) which were two magpies and a common buzzard (16).

There is little information describing the prevalence of N. caninum in birds found in Turkey. In a study conducted in Hatay province located in southern Anatolia and Van province located in eastern Anatolia, N. caninum Nc5 gene was detected in six wild avian species among 103 wild birds (16.5%) belonging to 20 different species (1).

In our study, Neospora positivity detected by PCR in wild birds was 5.45%. To our knowledge, this is the first report showing the presence of N. caninum in little tern in Turkey and the world. This result indicates that the wild birds can be important as intermediate hosts for N. caninum transmission according to the phylogenetic analysis results, the isolates obtained from birds were found to be close to each other. Also, according to Genbank data, it was found to be closely related to isolates obtained from Italy. The reason for this is that migratory birds can carry N. caninum strains to countries in the near geographic region.

This research was carried out in İzmir and Manisa provinces of Turkey which are one of the most important resting, hunting, feeding, and breeding areas on the migration routes of wild birds. These results indicate that natural water sources can be contaminated with N. caninum oocysts as well as small animals such as rodent or wild type mouse strains containing tissue cysts that may play an important role in transmission of N. caninum to wild birds. Moreover, since wild birds that are resistant to many pathogens can transmit neosporosis as well as other important zoonotic diseases such as avian influenza, toxoplasmosis, and Acanthamoeba keratitis to humans and animals, further epidemiological studies are required on this area (1,25,30-32).