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Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20

Molecular detection of Neospora caninum in house sparrows (Passer domesticus) in Iran ab

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Amir Abdoli , Mohsen Arbabi , Abdolhossein Dalimi & Majid Pirestani

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Department of Parasitology, Faculty of Medical Sciences, Kashan University of Medical Science, Kashan, Iran b

Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Accepted author version posted online: 14 May 2015.

Click for updates To cite this article: Amir Abdoli, Mohsen Arbabi, Abdolhossein Dalimi & Majid Pirestani (2015): Molecular detection of Neospora caninum in house sparrows (Passer domesticus) in Iran, Avian Pathology To link to this article: http://dx.doi.org/10.1080/03079457.2015.1050583

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Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology DOI: http://dx.doi.org/10.1080/03079457.2015.1050583

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Cavp-2015-0059.R1

Molecular detection of Neospora caninum in house sparrows (Passer

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Amir Abdoli 1,2, Mohsen Arbabi 1, Abdolhossein Dalimi 2, Majid Pirestani 2, 1

Department of Parasitology, Faculty of Medical Sciences, Kashan University of Medical 2

Modares University, Tehran, Iran.

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Science, Kashan, Iran. Department of Parasitology, Faculty of Medical Sciences, Tarbiat

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Running title: Neospora caninum in house sparrow.

Corresponding author: Mohsen Arbabi. Ph.D Email: [email protected] Tell: +98-9133611303

Tel:+98-31-555540021

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domesticus) in Iran

Fax:+98-31-555541112

Received: 4 April 2015

Abstract

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Neospora caninum is an intracellular protozoan parasite with a wide range of intermediate animal hosts. There is little information describing the prevalence and genetic characterization of N. caninum in bird hosts worldwide and in Iran. In this study, a total 217 brain samples of house sparrow (Passer domesticus) were examined for N. caninum presence by nested-PCR targeting

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the Nc-5 gene. Neospora caninum DNA was detected in 3.68% (8/217) of sparrows. Sequencing

of the Nc5 genomic DNA revealed 97-99% of similarity with N. caninum sequences deposited in Genbank. To our knowledge, this study is the first molecular evidence of N. caninum DNA in

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domestic environment.

Introduction

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domesticus) in maintaining and spreading N. caninum infection to canines in the feral and

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Neospora caninum is a cyst-forming apicomplexan parasite with a wide range of intermediate animal hosts (Dubey & Schares, 2011; Dubey, Schares, & Ortega-Mora, 2007). Neospora caninum was first reported as a parasite of the domestic dog that associated with encephalomyelitis and myositis (Bjerkås, Mohn, & Presthus, 1984), prior to description as a new genus and species Neospora caninum by Dubey and colleagues (Dubey, et al., 1988). Canines

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bird hosts in Iran. The results of this study highlight the role of the house sparrow (Passer

(such as dog, fox and wolf) are definitive hosts of N. caninum, and several mammalian species (such as sheep, cattle, and water buffalo) as well as birds serve as intermediated hosts (reviewed by (Dubey & Schares, 2011; Dubey, et al., 2007)). Neosporosis is an important cause of abortion in dairy cattle (Almeria & López-Gatius, 2013) with great economic losses (Reichel, et al.,

2013). Neospora caninum also infect a variety of wild animals, including wild birds (Gondim,

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2006). Ground-foraging birds like sparrows may be infected by N. caninum through ingestion of oocysts from the soil; hence these birds can act as reservoirs for N. caninum transmission to canines in the feral and domestic environment. Neospora caninum has been detected in several bird hosts including sparrows (Passer domesticus) (Gondim, et al., 2010), chickens (Gallus

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domesticus) (Gonçalves, et al., 2012), magpies (Pica pica) and buzzard (Buteo buteo) (Darwich,

et al., 2012). Recently, N. caninum antibodies were detected in 17.3% of 150 chickens from Iran (Sayari, et al., 2014) and 39.5% of 1324 chickens from several countries in the Americas (ranges

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Dubey, 2011). However, there is little information about molecular frequency (occurrence) of N. caninum in bird hosts worldwide and in Iran. Hence, the aim of this study was to use molecular detection to determine the occurrence of N. caninum in house sparrows (Passer domesticus) in

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Iran in one of the largest studies to be reported.

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Materials and methods

Study area. Tehran province is located to the north of the central plateau of Iran. Tehran is located at 34-36.5° longitude north and 50-53° longitude east and covers an area of about 12981 square kilometers (wikipedia, 2015). Tehran city has hot summers and moderate winters (ranging

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between 7.2% from United States of American to 83.6% from Nicaragua) (Martins, Kwok, &

between 28°-30°C from mid-July to mid-September and around 1°C from December-January), with an average annual rainfall of 200 millimeters (Farhadian, 2011).

Sample collection. A total of 217 house sparrows (Passer domesticus) were captured in different regions of Tehran from February 2013 to April 2014. All birds were captured in mist nets and

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killed by cervical dislocation; whole brains were removed and kept at -20°C until used. All animal experimentation protocols were approved by the Animal Care Committee of Tarbiat Modares University, Tehran, Iran.

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DNA extraction. Whole brains of each bird were homogenized in 20 ml phosphate-buffered

saline and DNA was extracted using a phenol-chloroform extraction method. Briefly, 800 μl of extraction solution (50 mM Tris-HCL, pH8.0; 25 mM EDTA and 400 mM NaCl), 100 μl 10%

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brain samples in 2 ml Eppendorf tubes. The suspension was incubated at 55ºC for 3 hours. For precipitation of undissolved debris and proteins, 300 μl 6 M NaCl was added to the suspension and kept at 4°C for 15 min, prior to centrifugation at 13,000 g for 15 min, after which the

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supernatants were transferred to new Eppendorf tubes (Biase, et al., 2002). Then, the samples were extracted with phenol–chloroform–isoamyl alcohol (25:24:1). DNA was precipitated by

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adding two volumes of cold 100% ethanol and 0.1 volume of sodium acetate solution (3M, pH 5.2), kept at -70°C for 60 min, followed by centrifugation at 13,000 g for 5 min. The pellet was washed twice with 70% ethanol and resuspended in 100 μl of distilled water. Concentration of DNA was determined by spectrophotometric analysis at 280/260 nm. The DNA was stored at -20 °C until used.

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SDS, and 30 μl proteinase K (20 μg/μl) (Fermentas, USA) were added to 80-100 μl homogenized

Nested-PCR . PCR was performed using a pair of N. caninum-specific primers Np21plus (5'CCCAGTGCGTCCAATCCTGTAAC-3') and Np6plus (5'CTCGCCAGTCCAACCTACGTCTTCT-3') (Müller, et al., 1996). Second round nested-PCR was performed with primers Np6 (5'-CAGTCAACCTACGTCTTCT-3') and Np7 (5'-

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GGGTGAACCGAGGGAGTTG-3') (Hughes, et al., 2006). The first round reaction was carried out in 20 μl reaction mixtures containing 10 μl 2 x master mixes (DFS Master Mix- BIORON GmbH), 10 pmol of each respective primer, 1 μl of template DNA and 7 μl of distilled water. Amplification of the first round reaction was conducted with initial denaturation for 5 minutes at

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94°C, followed by 40 cycles of 94°C for 40 seconds (denaturation), annealing at 62°C for 40 seconds, extension at 72°C for 40 seconds and final extension at 72°C for 10 minutes. The

second round nested-PCR was performed with the same reaction conditions as the first round;

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microlitre of the first round PCR product was used as the template for nested-PCR. For each reaction, a positive control (DNA extracted from the NC5 strain of N. caninum) and a negative control (double distilled water) were included. Five microliters of each PCR product was

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electrophoresed (by TAE buffer) through 1.5% (w/v) agarose gel stained with safe stain and

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visualized under ultra-violet trans-illumination.

Nucleotide sequence analysis. Three positive PCR products were extracted from the gel using a Vivantis Gel Purification kit (Vivantis, Malaysia) according to the manufacturer’s protocols. The nested-PCR products were directly sequenced in the forward and reverse directions by Sequetech Company (USA). The sequences were edited and manually checked with BioEdit Sequence

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except 25 pmol of primers (Np7 and Np6) and an annealing temperature of 56°C were used. One

Alignment Editor (Hall, 1999), aligned with Nc-5 partial sequences from other hosts by ClustalX

2.12 (Larkin, et al., 2007) and compared with sequences of N. caninum available in GenBank. Phylogenetic tree and evolutionary analyses were conducted using the neighbour-joining and maximum-likelihood model methods with MEGA6 software (Tamura, et al., 2013). The bootstrap scores were calculated for 1000 replicates.

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Results

Out of 217 house sparrows, presence of the Nc-5 gene was detected in eight birds (3.68%) (See

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supplementary figure 1). Three nucleotide sequences of the Nc-5 gene with a length of 227, 226 and 227 bp, respectively were submitted to GenBank database (GenBank accession nos.

KP702735, KP702736, KP702737). The results revealed our sequences shared 98-99% similarity

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joining method (Fig. 1) and Maximum Likelihood (supplementary figure 2) showed intraspecific variations among N. caninum isolates in this study and some other N. caninum deposited in GenBank. Analysis of Nc-5 sequence in one of our samples (KP702735) showed the highest

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similarity (100%) with N. caninum isolated from a wolf (KF649844), a chicken (EU073599) and 99% similarity with N. caninum from a wolf (KF649844, KF649845, KF649847, KF649848) and

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bison (HM031966). Our other sequences (KP702736, KP702737) showed 99% similarity with N. caninum isolated from a wolf (KF649844.1), chicken (EU073599.1), rat (DQ077662.1) and bison (HM031966.1) (Supplementary Table).

Discussion

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with each other and 97-99% similarity for N. caninum. Phylogenetic trees using neighbour

There is little information describing the prevalence of N. caninum in bird hosts worldwide or in Iran. In this regard, Sayari and colleagues previously detected anti-N. caninum antibodies in 17.3% of 150 free ranging chickens in southern Iran (Sayari, et al., 2014) but this study is the first molecular detection of N. caninum in bird hosts in Iran.

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There are some reports on the molecular occurrence of N. caninum detected in bird hosts worldwide. In this regard, Goncalves et al (Gonçalves, et al., 2012) detected N. caninum DNA in six out of 100 free ranging chickens in Brazil. Darwich and colleagues (Darwich, et al., 2012) defined presence of T. gondii and N. caninum DNA in brain samples from 14 species of wild

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birds from Spain. They diagnosed N. caninum DNA in three samples from 200 birds (1.5%),

including two magpies and one common buzzard (Buteo buteo). Toxoplasma gondii DNA was also detected in 12 samples (6%) (Darwich, et al., 2012). Gondim et al (Gondim, et al., 2010)

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after nucleotide sequencing (Gondim, et al., 2010). Serological study N. caninum in free-range chickens (Gallus gallus domesticus) revealed seroprevalence rates of 39.5% (ranging between 7.2% and 71.5%) from different countries in North and South America (Martins, et al., 2011).

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Several experimental and serological studies have also revealed that birds can be intermediated hosts for N. caninum (Costa, et al., 2008; Mansourian, et al., 2009; Martins, et al., 2011;

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McGuire, et al., 1999; Mineo, et al., 2009). Neospora caninum can induced several forms of pathologies in experimentally infected pigeons (Columba livia) (McGuire, et al., 1999; Mineo, et al., 2009), zebra finches (Poephila guttata) (McGuire, et al., 1999) and chickens (Mansourian, et al., 2009).

The prevalence of N. caninum infection in ground-foraging birds (like sparrows) can be

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detected Toxoplasmatinae DNA in 25% of 40 house sparrows with 7.5% found to be N. caninum

used to indicate environmental contamination with N. caninum oocysts. Also, ground-foraging birds can be potential reservoirs for N. caninum transmission to dogs and wild canids. Taken

together, the results of this study provide evidence that sparrows retain N. caninum in their tissues, and could potentially serve as a reservoir of infection to canines in the feral and domestic

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environment. However, more research is needed to reveal the role of birds in the epidemiology of neosporosis.

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Acknowledgements

We thank Dr. Javid Sadraei and Dr. Fatemeh Ghafarifar (from Tarbiat Modares University) for their sincere assistance throughout this study. The authors would like to thank Mr. Mahdi

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contributions made by two anonymous referees during critical review of the manuscript and their

Acknowledgements

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comments which were helpful in improving of our paper.

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This work was financially supported by Iran National Science Foundation (INSF) (grant No. 92019029).

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Rezapour for the help in preparation of the samples. The authors also appreciate the

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Figure 1. Phylogenetic relationships among Neopsora caninum based on a partial Nc5 gene sequence. The evolutionary history was inferred using the neighbour-joining method, supported by 1000 bootstrap replicates. Samples isolated in the present study (diamond dots) (KP702735, KP702736, KP702737) were compared to isolates selected from GenBank.

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Molecular detection of Neospora caninum in house sparrows (Passer domesticus) in Iran.

Neospora caninum is an intracellular protozoan parasite with a wide range of intermediate bird hosts. There is little information describing the preva...
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