Molecular survey of avian circoviruses in some non-psittacine birds and detection of a novel canary circovirus in a pigeon

Abstract

Background:

Circoviruses are small, non-enveloped, single stranded DNA viruses. There is scarce information about these agents in non-psittacine birds.

Aims:

It is attempted to detect and characterize circoviruses in non-psittacine birds.

Methods:

Forty-five samples were collected from different non-psittacine species belonging to seven avian orders. A nested polymerase chain reaction (nested-PCR) for the detection of rep gene of circoviruses was applied.

Results:

Two different types of circoviruses were detected in two pigeon samples (2/11, 18.2%). One of the detected circoviruses was placed in clade A next to a polish strain based on phylogenetic analysis. Interestingly, the other detected circovirus was closely related to canary circoviruses (CaCVs).

Conclusion:

In addition to the molecular diagnosis of a pigeon circovirus (PiCV), this is the first report of the detection of CaCv in a pigeon. The possible hypotheses of such circumstance are discussed.

Introduction

Circoviruses are small non-enveloped and icosahedral viruses with a circular, non-segmented, single stranded DNA. Circoviral agents convey two major open reading frames (ORFs); ORF V1 located in viral strand and encode the replication-associated proteins (Rep) and ORF C1, located in the complementary sense strand, encoding the viral capsid protein (Cap) (Todd, 2000 ▶). The family Circoviridae is composed of two genera including Cyclovirus and Circovirus. The genus Circovirus consists of 39 species. There were 11 known avian circoviruses. Beak and feather disease virus (BFDV), duck circovirus (DuCV), goose circovirus (GoCV), starling circovirus (StCV), canary circovirus (CaCV), pigeon circovirus (PiCV), swan circovirus (SwCV), raven circovirus (RvCV), zebra finch circovirus (ZfiCV), finch circovirus (FiCV), and gull circovirus (GuCV) are the established circoviruses (ICTV Virus Taxonomy, 2018). Because the virus is difficult to isolate (Todd, 2000), several features, including the host specificity are still debatable.

Most circoviruses such as SwCV, StCV, FiCV, and GuCV were discovered accidentally and associated with secondary infections (Todd, 2000 ▶; Johne et al., 2006 ▶; Halami et al., 2008 ▶). Pigeon circovirus infection is responsible for “young pigeon disease syndrome” (YPDS) (Raue et al., 2005 ▶). Circoviruses play a role in a condition called “Black spot” in neonatal canaries with distended abdomen and gall bladder congestion (Goldsmith, 1995).

In this study, a nested broad-spectrum polymerase chain reaction (nested-PCR) was used which should be capable of sensitive detection of a broad range of different circoviruses including circoviruses not currently known. According to the author’s knowledge, this is the first report of multi-species molecular survey of avian circoviruses in Iran.

Materials and Methods

Clinical samples were collected from non-psittacine birds in Tehran, Iran, during 2014. Samples were collected either individually (from faeces or triple sampling from conjunctiva, choanal cleft and cloaca) or mixed faeces from several birds with the same aviary bird species in three zoological collections. Blood samples and tissue specimens were collected individually from birds which have been referred to a veterinary clinic (Table 1). No anticoagulant was used for taking blood samples.

Table 1.

Clinical specimens from different avian species which were used for the molecular detection of avian circoviruses

Order	Species	Sample				Number of birds tested (total= 45)	Number of positive birds
		Blood	Triple swabsb	Faeces	Tissue
Columbiformes	Columba livia	2	7	2a	-	11	2 (Faeces, Blood)
Passeriformes	Serinus canaria	-	-	-	5	5	-
	Corvus cornix	-	-	-	2	2	-
	Acridotheres tristis	1	-	-	-	1	-
	Podoces pleskei	-	-	1	-	1	-
	Pyrrhocorax pyrrhocorax	-	-	1	-	1	-
Anseriformes	Mixed species of ducksc	-	-	3a	-	3	-
	Mixed species of swansc	-	-	2a	-	2	-
	Mixed species of geesec	-	-	3a	-	3	-
	Anas platyrhynchos	1	-	-	-	1	-
	Tadorna ferruginea	-	1	-	-	1	-
	Cairina moschata	-	-	2a	-	2	-
	Cygnus olor	-	-	-	1	1	-
	Cygnus atratus	-	-	-	1	1	-
Galliformes	Meleagris gallopavo	-	-	-	3	3	-
	Pavo muticus	-	-	-	1	1	-
Phoenicopteriformes	Phoenicopterus roseus	-	-	1a	-	1	-
Struthioniformes	Struthio camelus	-	-	-	1	1	-
Gruiformes	Balearica regulorum	-	-	4	-	4	-

^a Each sample related to mixed faeces of several birds with same species that were kept in one cage, ^b The sample was taken with a single swab from conjunctiva, choana and cloaca respectively, and ^c Mixed species of the same genus kept in a single enclosure

The viral DNA was extracted using High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). A nested broad spectrum PCR using degenerated primers was applied for the detection of circoviruses (Halami et al., 2008 ▶). The primers targeted the rep gene with an expected size of 350 bps. Polymerase chain reaction products were evaluated using electrophoresis in a 2% agarose gel containing RedSafe ^TM (iNtRON BIOTECHNOLOGY, South Korea). The PCR products of the expected length were sequenced for confirmation (Fig. 1). Psittacine BFDV from the previous study was used as positive control DNA (Haddadmarandi et al., 2018 ▶).

Fig. 1.

Agarose gel electrophoresis of broad-spectrum degenerated PCR product targeting circovirus rep gene approximately 350 bp. Lanes 1-4: Negative samples, Lane 5: Pigeon sample, Lane 6: DNA ladder 100 bp, Lanes 7-12: Negative samples, Lane 13: BFDV positive sample from previous study. DNA smear and non-target faint band in Lane 5 are because of using degenerated broad spectrum primers. 350 bp DNA band was isolated from the gel and sequenced for confirmation

The positive products of the PCR were subjected to DNA sequencing by Bioneer Biotechnology (South Korea). Nucleotide sequences were submitted in GenBank (Table 2). For sequence analysis Basic Local Alignment Search Tool (BLAST), BioEdit (version 7.2.5) and MEGA 7 (Kumar et al., 2016 ▶) softwares were applied.

Table 2.

GenBank accession numbers of circovirus rep gene sequences detected in pigeons

Strain name	Host species	Accession No.
CaCV-MH-IR-Ch49-Rep-2014	Columba livia domestica	KU521520
CoCV-MH-IR-C12-Rep-2014	Columba livia domestica	KT962830

The isolate names were coded according to the scheme: ‘A’-MH-IR-‘B’-Rep-‘C’, where ‘A’ denotes the species of circovirus, MH refers to the name of the author (Mohammadreza Haddadmarandi), IR indicates the country (Iran), ‘B’ denotes the laboratory code, the Rep shows the replication part of circoviral genome, and the last ‘C’ shows the year of detection

Results

Forty-five samples belonging to seven orders including Anseriformes (14/45), Columbiformes (11/45), Passeriformes (10/45), Galliformes (4/45), Gruiformes (4/45), Struthioniformes (1/45), and Phoenicopteriformes (1/45) were collected (Table 1). Nineteen samples were taken from various referred bird species with a variety of clinical manifestations and the remaining were gathered from three different aviaries in Tehran named A1, A2 and A3.

Two samples were positive for circoviruses (4.4%). Both positive samples belonged to domestic pigeons (Columba livia). No characteristic clinical signs were seen in these two specimens. One of them which had been detected in the blood sample of a young pigeon was placed in clade A, next to Polish PiCV strain PL53 (GenBank accession No. Kf738860). Interestingly, the other circovirus was identified as CaCV (Fig. 2). It was detected in a group of pigeons housed in a single species pigeon loft.

Fig. 2.

Molecular phylogenetic analysis by maximum likelihood tree of rep gene partial sequences (350 bp) of different circoviruses. The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The Iranian circoviruses (IR) are marked with black square. All samples are named according to the scheme: Circovirus species (strain name) - (GenBank accession number) host species followed by - Columbid circovirus geno-group, if applicable

Discussion

In the present study, two types of circoviruses were detected in pigeons using a previously established nested broad-spectrum PCR (Halami et al., 2008 ▶). The characterization of detected circoviruses was performed using the partial sequencing of rep gene.

Pigeon circoviruses could be genetically grouped in five different genogroups (A-E). The reported infection rates were 57% in Hungary (Cságola et al., 2012 ▶), 32% in Belgium (Hattermann et al., 2002 ▶), 48% in Germany (Soike et al., 2001 ▶), 70% in Poland (Stenzel and Pestka, 2014 ▶), and 75% in China (Zhang et al., 2015 ▶). The first report of PiCVs in Iran showed the prevalence of 24% of PiCV in Chaharmahal va Bakhtiari province (Mahzounieh et al., 2014 ▶). The only positive PiCV of the present study placed with those classified as group “A” PiCVs were closely related to the strain which was detected in Poland (Stenzel et al., 2014 ▶). According to the limited sample size in the present study, it is not possible to show the prevalence of PiCV infection in Tehran.

The other positive sample was detected a single species aviary which only pigeons were kept in. It was identified as CaCV. As the aviary was not a completely closed enclosure, contamination of the specimen with canary or other wild bird droppings was also possible. Circoviruses are highly resistant to chemical and physical agents (Todd, 2000 ▶) and therefore can survive for a long time in the environment or even gastrointestinal tract of the pigeon. Such assumption was previously reported as StCV was detected in an Estuarine Mollusc (Amphibola crenata) as a passenger (Dayaram et al., 2013 ▶). There is a debate in host specificity among circoviruses and very few studies have been performed on CaCV (Phenix et al., 2001 ▶; Todd et al., 2001 ▶; Rampin et al., 2006 ▶). Hence the host specificity of the virus remains uncertain. The detected CaCV has only 98% identity with other CaCV in the GenBank, so it could be an intermediate strain which might pass the host barrier. A self-limiting viral host-switching of BFDV between Psittaciformes and Coraciiformes has recently been shown (Sarker et al., 2015 ▶). There could be high mutation rate within circoviruses (Halami et al., 2008 ▶) and these mutations can possibly change the host specificity of circoviruses. Another hypothesis for the presence of the canary-like circovirus in a pigeon sample could be the viral genome mutations or recombinations (Bassami et al., 2001 ▶; Rau et al., 2004 ▶; Hughes and Piontkivska, 2008 ▶). Full genome sequencing is needed to determine the potential capability of these viral changes. Unfortunately, it was not possible to find the exact positive pigeon in the aviary to re-sample and re-examine it for the presence of circoviruses.

Circoviruses were detected in the present study in clinical specimens of pigeons. Further studies with larger sample sizes from different geographical regions are needed to elaborate host specificity and the incidence of the infection in the country.