Pathology and phylogenetic analysis of some viruses identified in wildlife birds in croatia: preliminary research



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PATHOLOGY AND PHYLOGENETIC ANALYSIS OF SOME VIRUSES IDENTIFIED IN WILDLIFE BIRDS IN CROATIA: PRELIMINARY RESEARCH
Marina Tišljar1, Marina Biđin2, Zdenko Biđin2, Vladimir Savić1, Borka Šimpraga1, Tajana Amšel Zelenika1
1Croatian Veterinary Institute, Poultry Centre, Zagreb, Croatia

2Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
Marina Tišljar, DVM, PhD, Poultry Centre, Croatian Veterinary Institute, 10000 Zagreb, Croatia; tel: +385 (0)1 2440 214; fax: +385 (0)1 2441 396; e-mail: tisljar@veinst.hr
Summary

The article documents the results of pathomorphological and molecular diagnostic testing (polymerase chain reaction, PCR) for adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus determination in a sample of 16 wildlife birds of nine various species (white stork (Ciconia ciconia), mute swan (Cygnus olor), Caspian gull (Larus cachinnans, Pallas 1811), mallard duck (Anas platyrhynchos), common buzzard (Buteo buteo), white-tailed eagle (Haliaeetus albicilla), domestic pigeon (Columba livia domestica), barn swallow (Hirundo rustica) and sparrow (Passer sp.). The birds were necropsied at the Laboratory of Pathology, Poultry Centre, Croatian Veterinary Institute, Zagreb, Croatia. Dual infections with astrovirus and circovirus were confirmed in mallard duck having died of the consequences of botulism. The sequence DuCV/M10-448 derived from the mallard duck samples had the greatest nucleotide (99%) and amino acid (100%) similarities with duck circovirus (accession number DQ100076.1) detected in the USA. The PCR positive result of hepadnavirus infection was partly confirmed by histopathologic examination in Caspian gull which suffered from generalized amyloidosis. According to the authors' awareness, this was the very first finding of hepadnavirus in gulls. The circovirus sequence found in the domestic pigeon (PiCV/M10-513) showed a 100% nucleotide and amino acid homology with pigeon circovirus (accession number AF252610.1) identified in Germany. In one of virologically tested mute swans, pathomorphological examination confirmed iron storage disease (ISD), while the rest of the birds died of the consequences of ballistic trauma or gunshot wounds (GSW) and other traumatic lesions (white stork, mute swan, common buzzard, white-tailed eagle, barn swallow and sparrow). Molecular diagnostic test results for the West Nile, avian influenza and Newcastle disease viruses were negative.


Key words: wildlife birds, pathomorphology, histopathology, polymerase chain reaction, astrovirus, hepadnavirus, adenovirus, circovirus, coronavirus, Caspian gull (Larus cachinnans, Pallas 1811), mallard duck (Anas platyrhynchos), domestic pigeon (Columba livia domestica), mute swan (Cygnus olor), iron storage disease (ISD), amyloidosis
Introduction

Viruses from the genus Aviadenovirus, and Atadenovirus from the Adenoviridae family are known to infect domestic poultry and are described in several wild bird species, i.e. mallard duck, gulls, psittacines, owls, hawks, pigeons, ostriches and other avian species (McFerran and Smyth, 2000; Harrach and Kaján, 2011). Adenovirus infection has been widely investigated in domestic poultry, while there are not much data available on the infection and outcome of the disease in various wild bird species. Avian astroviruses are small (27-30 nm in size) positive sense RNA viruses, with the genome length of approximately 7 kb. While astrovirus infection has been widely investigated in domestic poultry (enteric disease in chickens and turkeys, and fatal hepatitis in ducklings), little is known about the clinicopathologic manifestations of astrovirus infection in free-living birds. Recently, astroviruses have been identified in guinea fowl, pigeons, domestic geese and several duck species, suggesting that astroviruses are widespread among avian hosts (Schultz-Cherry and Lorne, 2013). Circoviruses are spherical and single-stranded (ssDNA) viruses with a genome of approximately 2 kb. They are currently classified into two genera, Circovirus and Gyrovirus, both found to infect birds. Generally, circovirus infection results in immunosuppression accompanied by symptoms ranging from anemia and lymphoid atrophy in chickens, feather abnormalities in psittacines, diarrhea, ill-thrift in pigeons, to growth retardation in ducks (Todd, 2004). Par and Robert (2007) report on gull circovirus, goose circovirus, Laughing (Senegal) Dove circovirus, ostrich circovirus and canary circovirus. These authors also mention identification of avian circovirus in a spectrum of avian species encompassing psittacines, Columbidae, passerines (Estrildidae, Fringillidae), Anatidae, Phasianidae and Struthionidae. Recently, new types of circoviruses have been identified in some mammalian species and chickens (Li et al., 2010). Avian hepadnaviruses are DNA viruses that have been primarily identified as the causative agents of hepatitis B in ducks. They are hepatotropic viruses with a very narrow host range (Schödel et al., 1989). So far, they have been described in a variety of domestic and exotic birds, including domestic ducks and geese, Mandarin duck, Ross’s goose, snow goose, ashy-headed sheldgoose, crane, heron, stork and parrot (Woolcock and Tsai, 2013). Since duck hepatitis B virus (DHBV) has been used as a common model for HBV infection in humans (Mason et al., 1980), the pathogenesis and histopathologic changes associated with DHBV infection have been widely investigated. Unlike that, other known avian hepadnaviruses have been characterized mostly upon their genomes. Coronaviruses are enveloped RNA viruses with a positive-strand genome, and have been associated with diseases in several mammalian species and birds. In poultry, coronaviruses cause infectious bronchitis in chickens and enteric disease in turkeys, while they have also been identified in peafowl, teal and racing pigeons (Monceyron Jonassen et al., 2005). Hughes et al. (2009) detected coronavirus in wildfowl (Anseriformes) and waders (Charadriiformes), and Chu et al. (2011) detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds (Anseriformes, Ciconiiformes and Pelecaniformes birds).

A total of 231 wildlife birds of various species (pigeons /Columbidae/, pheasants and grouse /Phasianidae/, birds of prey (eagles /e.g., white-tailed eagle - Haliaeetus albicilla, golden eagle - Aquila chrysaetos; hawks /Sparrowhawk - Accipiter nisus/, vultures /e.g., griffon vulture - Gyps fulvus/; owls (Ural Owl - Strix uralensis /Strigidae/), buzzard (e.g., common buzzard - Buteo buteo (Accipitridae); wading birds (e.g., white stork /Ciconiidae/); swans and ducks (e.g., mute swan - Cygnus olor and mallard duck - Anas platyrhynchos/Anatidae/); gulls (e.g., Caspian gull - Larus cachinnans, Pallas 1811, European herring gull - Larus argentatus, Black-headed gull - Chroicocephalus ridibundus /Laridae/); passerine birds (e.g., great tit - Parus major /Paridae/, Euroasian tree sparrow - Passer montanus (Passeridae/, barn swallow - Hirundo rustica /Hirundinidae/; Sylvia Communis /Sylviidae/, etc.); woodpeckers (great spotted woodpecker – Dendrocopos maior /Picidae/; blackbird (common blackbird - Turdus Merula /Turdidae/, etc.) were routinely necropsied at the Laboratory of Pathology, Poultry Centre, Croatian Veterinary Institute, Zagreb, Croatia during the 2010-2014 period.

Since there are not much available data on adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus infections, outcome and clinicopathologic manifestations of the infection in free-living birds, preliminary pathomorphology and molecular biology investigations of the adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus presence were carried out in a randomly chosen sample of 16 wildlife birds of nine various bird species (white stork (Ciconia ciconia), mute swan (Cygnus olor), Caspian gull (Larus cachinnans, Pallas 1811), mallard duck (Anas platyrhynchos), common buzzard (Buteo buteo), white-tailed eagle (Haliaeetus albicilla), domestic pigeon (Columbia livia domestica), barn swallow (Hirundo rustica) and sparrow (Passer sp.).


Material and Methods

Case description and sampling methods

Routine necropsy procedures of the total of 231 wildlife birds were performed during the 2010-2014 period at the Laboratory of Pathology, Poultry Centre, Croatian Veterinary Institute, Zagreb, and, where indicated, histologic, virologic, bacteriologic and other diagnostic techniques (x-ray examination) were applied. Only the birds examined in 2014 were tested for West Nile virus infection and the birds necropsied during the 2010-2014 period were tested for avian influenza and Newcastle disease virus infections (Laboratory of Virology, Poultry Centre, Croatian Veterinary Institute, Zagreb, Croatia). The organs dissected from 16 birds of nine various species (white stork (Ciconia ciconia), mute swan (Cygnus olor), Caspian gull (Larus cachinnans, Pallas 1811), mallard duck (Anas platyrhynchos), common buzzard (Buteo buteo), white-tailed eagle (Haliaeetus albicilla), domestic pigeon (Columba livia domestica), barn swallow (Hirundo rustica) and sparrow (Passer sp.)) were tested using a molecular biology testing procedure for adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus infections (Department of Poultry Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, Croatia).


Pathomorphology analysis

During necropsy, tissues were taken for subsequent histopathologic, virologic, bacteriologic and other diagnostic examinations. For histopathologic analysis, organ samples were fixed in neutral 10% formalin solution, embedded in paraffin and cut into 4-m thick sections on a rotary microtome (MICROM HM 325; Zeiss, Austria). After deparaffinization, the sections were stained with hematoxylin and eosin (HE) using Congo-red and Prussian Blue techniques for confirmation of amyloid and of iron. The slices were examined under the light microscope (LEICA DMLB, Germany) and the images were captured with a PIXERA Pro 150ES digital camera.


Molecular diagnostic testing for adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus determination

Molecular diagnosis

The organs were dissected and collected for molecular diagnostic testing (Table 1). DNA and RNA were extracted in a volume of 50 L from organ homogenates using High Pure Viral Nucleic Acid Kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). DNA was synthesized using SuperScript TM III (Invitrogen, Carlsbad, California, USA). Reverse transcription reaction (42 oC for 50 min and inactivation for 15 min at 70 oC) was carried out in the Gene AMP PCR Systems 2400 (California, USA). PCR reaction: 1.5 L of DNA or cDNK, 25 L of Go Taq Green Master Mix (Promega, Madison, USA) and 0.20 M forward and reverse primer contained in a total volume of 50 L. The PCR reaction conditions were adjusted according to the references shown in Table 2. The PCR reaction products were analyzed by 1.5% gel electrophoresis stained with ethidium bromide.


Table 1. Alphabetical list, scientific name, year of sampling, laboratory identifier and collected organs of birds tested
Tablica 1. Abecedni popis i latinski naziv, godina prikupljanja, laboratorijska oznaka i uzorkovani organi ptica


Bird species

Scientific name

Year

Identifier

Organs

White stork

Ciconia ciconia

2013

M13-69

Intestine, liver, spleen, kidney

Mute swan

Cygnus olor

2011

M11-794

Intestine, liver

Mute swan

Cygnus olor

2013

M13-82

Intestine, kidney

Mute swan

Cygnus olor

2014

M14-01

Intestine, liver

Mallard duck

Anas platyrhynchos

2010

M10-448

Intestine, spleen, bursa of Fabricii

Domestic pigeon



Columba livia domestica

2010

M10-513

Liver, kidney

Barn swallow

Hirundo rustica

2012

M12-906

Intestine, liver, spleen, kidney

Caspian gull



Larus cachinnans, Pallas 1811

2012

M12-950

Liver, kidney

Common buzzard

Buteo buteo

2011

M13-54

Intestine, liver, spleen, kidney

Common buzzard

Buteo buteo

2013

M13-55

Intestine, liver, spleen, kidney

Common buzzard

Buteo buteo

2013

M13-56

Intestine, liver, spleen, kidney

Common buzzard

Buteobuteo

2013

M13-57

Intestine, liver, spleen, kidney

Common buzzard

Buteo buteo

2013

M13-58

Intestine, liver, spleen, kidney

Common buzzard

Buteo buteo

2013

M13-59

Intestine, liver, spleen, kidney

White-tailed eagle

Haliaeetus albicilla

2011

M12-792

Intestine, liver, spleen, kidney

Sparrow

Passer sp.

2012

M 12-907

Intestine, liver, spleen, kidney



Sequence analysis

The samples showing the expected amplicon length were purified using QI Aquick PCR Purification Kit (Qiagen, Germany) and sequenced in Macrogen Inc. (Amsterdam, the Netherlands). The virus sequences were initially checked against BLAST search database (http:/www.ncbi.nlm.nih.gov/BLAST/). The sequences were assembled and analyzed using Bio Edit (Hall, 1999), Mega 6 (Tamura et al., 2013) and Staden (Bonfield et al., 1995). Phylogenetic analysis was performed using Mega 6, while the best-fit phylogenetic model was selected using Model Test 2 (Darriba et al., 2012) (Table 2). The Gen Bank (http://www.ncbi.nlm.nih.gov/genbank/) accession numbers of the circovirus sequences submitted through this study were KP773230 (sequence PiCV/M10-513) and KP773231 (sequence DuCV/M10-448), while the accession numbers of sequences retrieved from the Gen Bank, shown in Figure 1.


Table 2. Oligonucleotide sequences, target gene, PCR amplicon size and references of primers used in the study

Tablica 2. Sekvence, ciljni gen, veličina proizvoda PCR i referenca početnica korištenih za pretraživanje ptica na navedene viruse


Virus

Primers

Target gene

Amplicon size

Reference

Adenovirus

F1: 5'-TNMGNGGNGGNMGNTGYTAYCC-3'

Polymerase

330 bp

Wellehan et al., 2004

R1: 5'-GTDGCRAANSHNCCRTABARNGMRTT-3'

F2: 5'-GTNWYGAYATHTGYGGHATGTAYGC-3'

R2:5'-CBCDRTTRTGNARNGTRA-3'

Astrovirus

F: 5'-GAYTGGACIMGITAYGAYGGIACIATICC-3'

Polymerase

434 bp

Todd et al., 2009

R: 5'-YTTIACCCACATICCRAA-3'

Circovirus

F1: 5'-GGIAYICCIAYYTICARGG-3'

Replication gene

400 bp

Li et al., 2010

R1: 5'-AWCCAICCRTARAARTCRTC-3'

F2: 5'-GGIAYICCICAYYTICARGGITT-3'

R2: 5'-TGYTGYTCRTAICCRTCCCACCA-3'

Hepadnavirus

F: 5'-CTCAAGAGATTCCTCAGCC-3'

Polymerase

906 bp

Walters et al., 2004

R: 5'-GTCATACCATTCTCCTACT-3'

Coronavirus

F: 5'-TCACAYTTWGGATARTCCCA-3'

Replication gene

250 bp

Stephenson et al., 1999



Molecular diagnostic testing for West Nile, avian influenza and Newcastle disease virus determination

RNA extraction

Tissue samples were homogenized in the equal volume of sterile buffered saline. The homogenates were centrifuged at 2000 g for 20 minutes and the resulting supernatant was used for RNA extraction. RNA extraction was done manually from the supernatants using High pure Viral Nucleic Acid Kit (Roche Applied Science, Germany), as described in the kit protocol.


Reverse transcription and real time PCR

One-step reverse transcription followed by real time PCR was carried out in LightCycler 1.5 thermocycler using LightCycler RNA Master HybProbe (Roche Applied Science, Germany). For the detection of West Nile virus, avian influenza virus, Newcastle disease class I as well as class II viruses, protocols according to Tang et al. (2006), Spackman et al. (2002), Wise et al. (2004) and Kim et al. (2008) were used, respectively.



Bacteriology and mycology analyses


Salmonella spp. isolation

Salmonella was isolated from the organs following the instructions for the standard EN ISO 6579:2002 and IOE methods (2008).


Isolation of the genus Streptococcus

All the analyzed organs were trans-inoculated onto the blood agar (Columbia agar with the supplementation of 5%-10% of sheep blood), Columbia agar and MacConkey agar. Streptococci did not grow in MacConkey agar (Wages, 2003).


Isolation of the genus Staphylococcus

All the investigated organs were trans-inoculated onto the blood agar (Columbia agar with the supplementation of 5%-10% of sheep blood), Columbia agar and MacConkey agar. Simultaneously, the organs were trans-inoculated onto mannitol salt agar and onto Baird-Parker agar, these representing selective culture media for Staphylococcus isolation. Staphylococci did not grow in MacConkey agar (Andreasen, 2003).


Escherichia coli isolation

At isolating the bacterium Escherichia coli, all the analyzed organs were trans-inoculated onto the blood agar (Columbia agar with the supplementation of 5%-10% of sheep blood), Columbia agar and MacConkey agar. Simultaneously, the organs were trans-inoculated onto the TBX agar, a selective medium for E. coli isolation (Barnes et al., 2003)


Clostridium spp. isolation

All the analyzed organs were trans-inoculated onto the blood agar (Columbia agar with the supplementation of 5%-10% of sheep blood), and incubated in an anaerobic environment (Barnes, 2003).


Fungi and molds isolation

Sabouraud glucose agar was used for the isolation of fungi and molds (Kunkle, 2003).


Shigella isolation

All the analyzed organs were trans-inoculated onto the blood agar (Columbia agar with the supplementation of 5%-10% of sheep blood), Columbia agar and Mac Conkey agar, and incubated in an aerobic environment (Naglić et al., 2005)

Biochemistry characterization for all the isolated bacteria was performed using API System strips.
Results

Pathomorphology investigation
Table 3. Alphabetical list, scientific name, year of necropsy, laboratory identifier and pathomorphological findings in birds tested for adenovirus, astrovirus, hepadnavirus, coronavirus and circovirus in the present study
Tablica 3. Abecedni popis, latinski naziv, godina patoanatomske pretrage, laboratorijska oznaka (virusa) i patomorfološki nalazi ptica pretraživanih na prisutnost adenovirusa, astrovirusa, hepadnavirusa, koronavirusa i cirkovirusa


Bird

species



Scientific

name



Year


Identifier


Pathoanatomical (PA) diagnosis/findings

Histopathologic (HP)

and/or x-ray analysis

(diagnosis/findings)


White stork



Ciconia ciconia



2013



M13-69



Gunshot wound (GSW) (frontal part of head)

X-ray findings of lead bullets (no HP investigation)

Mute swan

Cygnus olor

2011

M11-794

Gunshot wounds (GSW) (left wing and ribs with internal hemorrhages)

X-ray findings of lead bullet particles (no HP investigation)

Mute swan



Cygnus olor



2013



M13-82



Iron storage disease (ISD)


Iron pigment in hepatocytes (Prussian Blue staining) and focal necroses in the liver

Mute swan

Cygnus olor

2014

M14-01

Gunshot wounds (GSW)

X-ray analysis

(lead bullet particles)



Mallard* duck

Anas platyrhynchos

2010

M10-448

Type C botulism

Adenovirus-like and circovirus inclusions in intestine

Domestic** pigeon

Columba livia domestica

2010

M10-513

Sepsis

Focal mononuclear cell myocarditis; intra-cytoplasmic inclusions in hepatocytes

Barn swallow

Hirundo rustica

2012

M12-906

Traumatic wounds (head trauma)

No HP investigation

Caspian*** gull

Larus

cachinnans,

Pallas 1811



2012

M12-950

Icterus, hepatomegaly, proventricular hyperplasia

Generalized amyloidosis (Congo-red staining for amyloid)

Common buzzard

Buteo buteo

2011

M13-54

Traumatic wounds (head trauma)

No HP investigation

Common buzzard

Buteo buteo

2013

M13-55

Traumatic wounds (wing, leg and ribs)

No HP investigation

Common buzzard

Buteo buteo

2013

M13-56

Traumatic wounds (head, neck)

No HP investigation

Common buzzard

Buteo buteo

2013

M13-57

Gunshot wounds (GSW)

X-ray analysis (lead bullet particles)

Common buzzard

Buteo buteo

2013

M13-58

Traumatic wounds (legs)

No HP investigation

Common buzzard

Buteo buteo

2013

M13-59

Traumatic wounds (head, neck)

No HP investigation

White-tailed eagle

Haliaeetus

albicilla

2011

M12-792

gunshot wounds (GSW)

X-ray analysis (lead bullet particles)

Sparrow

Passer sp.

2012

M 12-907

Traumatic wounds (head)

No HP investigation

*dual infections with astrovirus and circovirus (mallard duck /Anas platyrhynchos/)

**circovirus infection (domestic pigeon /Columba livia domestica/)

***hepadnavirus infection (Caspian gull /Larus cachinnans, Pallas 1811/)





Molecular diagnostic testing for adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus determination

PCR assays and sequence analysis

Positive PCR results are shown in Table 4. The circovirus sequence found in pigeon (PiCV/M10-513) showed a 100% nucleotide and amino acid homology with pigeon circovirus (accession number AF252610.1) identified in Germany. The sequence DuCV/M10-448 derived from the mallard duck samples had the greatest nucleotide (99%) and amino acid (100%) similarities with duck circovirus (accession number DQ100076.1) detected in the USA. The sequence shared the greatest nucleotide (99%) and amino acid (100%) similarities with duck circovirus (accession number DQ100076.1) detected in the USA. The phylogenetic analysis results were consistent with those of molecular analysis, and clustered the sequence PiCV/M10-513 within the group of pigeon circoviruses, while the sequence DuCV/M10-448 was grouped together with duck circoviruses (Figure 1).


Table 4. Positive PCR results for one or more viruses

Tablica 4. Ptice pozitivne na jedan ili više pretraživanih virusa


Identifier

Host species

Virus

Organ

M10-448

Mallard duck

Astrovirus

Intestine, spleen

Circovirus

Intestine, spleen, bursa of Fabricii

M12-950

Caspian gull

Hepadnavirus

Liver

M10-513

Domestic pigeon

Circovirus

Liver, kidney

PCV-1 (KJ408798.1) pig



PiCV/M10-513 domestic pigeon

CoCV (AF252610.1) pigeon

CoCVPL60 (KF738863.1) pigeon

PiCV/Belgium/11-07574/2011 (JX901127) pigeon

PiCV (DQ915959.1) dove

StCV (DQ172906.1) starling

StCV AM-C (KC846095.1) starling

BFDV (AF080560.1) cockatoo

BFDV (AF311296.1) lovebird

GuCV (DQ845074.1) gull

GuCV (JQ685854.1) gull

RaCV (DQ146997.1) raven

CaCV (DQ339095.1) canary

CaCV (AJ301633.2) canary

FiCV (DQ845075.1) finch

GoCV (AF536934.1) goose

GoCV xs4 (DQ192285.1) goose

SwCV Sw4 (EU056310.1) swan

SwCV H51 (EU056309.1) swan

DuCV FJPT60 (GQ423746.1) Muscovy duck

DuCV (HQ180265.1) Muscovy duck

DuCV (AY228555.1) mulard duck

DuCV D12-KD-028 (KC851821.1) domestic duck

DuCV/M10-448 mallard duck

DuCV 33753-52 (DQ100076.1) Pekin duck

100

100


100

99

100



99

98

80



71

98

72



82

93

90



100

92

0.2


Figure 1. Phylogenetic Neighbor-Joining (NJ) tree based on alignments of the partial (417 nt) fragment of 26 circovirus replication gene sequences. The tree was constructed using JukesCantor model. Circovirus sequences identified in this study are highlighte d in grey. Porcine circovirus was used as an outgroup. Bootstrap values (>70) were shown next to the branches. The scale on the bottom represents evolutionary distances of the compared sequences.
Slika 1. Filogenetsko Neighbor-Joining (NJ) stablo dobiveno analizom nukleotidnih sekvenci replikacijskog gena cirko virusa dužine 417 nukleotida. Korišten je JukesCantorov (JC) model odabran prema analizi BIC (engl. Bayesian Information Criterion). U analizu je uvršteno ukupno 26 sekvenci iz roda Circovirus. Sekvence dokazane u ovom radu označene su žutom sivom bojom. Cirko virus svinje je korišten kao tzv. vanjska skupina (engl. outgroup). Vrijednosti bootstrap probe (>70) označene su uz ogranke stabla. Ljestvica na dnu slike označava evolucijsku udaljenost uspoređivanih sekvenci.
Molecular diagnostic testing for West Nile, avian influenza and Newcastle disease virus determination

Molecular diagnostic testing for West Nile virus, avian influenza and Newcastle disease virus determinations did not reveal positive results in any of the tested samples.



Bacteriology and mycology analyses


The most severe mixed microbial infections were determined in the domestic pigeon (Columba livia domestica) infected with circovirus (E. coli, Shigella sp., Aspergillus fumigatus, and molds) and in mallard ducks (Anas platyrynchos) infected with astrovirus and circovirus (E. coli, Clostridium sp., Micrococcus sp., Bacillus sp.).
Discussion

In 12 out of 16 wildlife birds selected for preliminary virologic testing for the presence of adenovirus, astrovirus, circovirus, hepadnavirus and coronavirus, ballistic and/or traumatic lesions of various origin were confirmed by pathoanatomical and x-ray examinations (Table 3). In the mute swan (Cygnus olor) (M 13-82) (Table 3), the gross findings of hepatomegaly with multiple dark foci in gold-brown liver aroused suspicion of iron storage disease (ISD). The histopathology examination confirmed ISD using special histologic staining for iron pigment, Prussian Blue staining. The ISD is one of pigmentary hepatopathies developing due to iron accumulation in hepatocytes. It is predominantly a disease of captive mynahs (Gracula sp.), several species of toucans (Ramphastide) and birds of paradise (Paradisaeidae). Infrequently, it can be seen in other bird species. The most susceptible are birds highly efficient at absorbing dietary iron and not down regulating iron absorption sufficiently when fed iron-rich diets. However, in wild birds it is presumed that iron is in a form that is not readily absorbed from the digestive tract (Schmidt et al., 2003). Our pathomorphological findings (gold-brown liver with dark spots and the histopathologic findings of iron pigment in hepatocytes and Kupffer's cells) confirmed ISD and not just the excessive liver iron in this swan. Dual infection with astrovirus and circovirus was confirmed in the mallard duck (Anas platyrhynchos) died from botulism (Clostridium botulinum toxin type C) (Tables 3 and 4). Bacteriologic investigations were positive for Clostridium sp. in the gizzard, the most common site of Clostridium botulinum because of the anaerobic environment in the gizzard cuticle. Type C botulism can also be caused by the ingestion of a preformed toxin. Outbreaks in waterfowl and other water birds are the result of the consumption of toxin-laden invertebrates (Dohms, 2003). Histopathology findings of the inclusion typical of circovirus infection were shown in one intestinal sample (Table 3). In another duck, however, the adenovirus-like inclusions were confirmed in the liver and intestine. Since one of the most prominent characteristics of circovirus infection is its immunosuppressive effect, it may be possible that simultaneous (adeno)virus infection was present.



According to the authors’ awareness, the hepadnavirus determined in the Caspian gull (Laruscachinnans, Pallas 1811) (Table 4) represents the very first finding of this virus in gulls so far. Generalized amyloidosis was confirmed by the special Congo-red staining for amyloid (Table 3), and –, according to Zschiesche and Jakob (1989) – , it represents a common finding in Laridae. Avian amlyoidosis can affect a wide variety of birds and is found following a number of diseases, especially chronic infections and inflammation. So, the simultaneous findings of hepadnavirus infection and amyloidosis in the Caspian gull (Tables 3 and 4) may arouse suspicion that generalized amyloidosis was the consequence of chronic hepadnavirus infection. Avian amyloidosis is normally a well-recognized pathologic disorder in birds, especially in the waterfowl (Landman et al., 1998).

In the domestic pigeon (Columba livia domestica), circovirus presenting with gross lesions of sepsis and mycotic inflammation (E. coli, Shigella sp. and Aspergillus sp., and molds, respectively) (Table 3) were determined as well (Table 4). Atypical intracytoplasmic inclusions were found in the liver sample, but no typical circovirus inclusions were seen in immunocompetent organs. Schmidt et al. (2003) compared this type of inclusions with atypical adenovirus inclusions. In pigeons infected with circovirus, Abadie et al. (2001) also found lesions that could have been related to bacteria, but with no histologic evidence of concomitant viral infection (e.g., adenoviral and/or herpesviral infections).
Conclusions

In conclusion, although this study was performed on a limited amount of samples, the findings of astrovirus, circovirus and hepadnavirus suggest that these viruses are widespread among avian hosts. Based on the molecular and phylogenetic analyses of circovirus sequences identified in the pigeon and in the mallard duck, it may be concluded that the same types of viruses circulate and can be found in different geographical regions. However, due to the possible limitations of PCR (e.g., sensitivity of primers), different levels of virus amount in the samples and the stage of autolysis of the collected organs, some viruses could have remained undetected using the current procedures. For that reason, further research combining molecular methods and histopathologic examination is needed to get deeper insight into the free-living bird diseases and pathology.


Acknowledgment

The research was supported by grant 053-0531863-1856 from the Ministry of Science, Education and Sports, Republic of Croatia.



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Bolesti i filogenetska analiza nekih virusa identificiranih u slobodno živućih ptica u Hrvatskoj: Početno istraživanje
Sažetak

U radu su prikazani rezultati patološkomorfološke pretrage i pretrage metodama molekularne biologije (lančana reakcija polimerazom /PCR/) za izdvajanje dokaz adenovirusa, astrovirusa, cirkovirusa, hepadnavirusa i koronavirusa u devet različitih vrsta sveukupno 16 slobodnoživućih ptica (bijela roda (Ciconia ciconia), crvenokljuni labud (Cygnus olor), galeb klaukavac (Larus cachinnans, Pallas 1811), divlja patka (Anas platyrhynchos), obični škanjac (Buteo buteo), orao štekavac (Haliaeetus albicilla), domaći golub (Columba livia domestica), lastavica pokućarka (Hirundo rustica) i vrabac (Passer sp.)). Ptice su razuđene u Laboratoriju za patologiju Centra za peradarstvo, Hrvatski veterinarski institut, Zagreb. U divlje patke (Anas platyrhynchos) uginule od posljedica botulizma (C toksin Clostridium botulinum) izdvojeni dokazani su cirkovirus i astrovirus. Niz Cirkovirus nukleotidnog slijeda DuCV/M10-448 izdvojen izdokazan u divlje patke pokazivao je najveću nukleotidnu (99%) i amino kiselinsku sličnost (100%) s pačjim cirkovirusom (pristupni broj DQ100076.1) utvrđenim u SADama. Pozitivni rezultat pretrage metodom PCR na izdvajanjeza dokaz hepadnavirusa u galeba klaukavca (Larus cachinnans, Pallas 1811), uginulog od posljedica amiloidoze potvrđen je djelomice i patološkohistološkim nalazom. Prema saznanju autora, ovo je ujedno prvi nalaz hepadnavirusa u galebova. Cirkovirusni niz utvrđen u domaćeg goluba (PiCV/M10-513) pokazao je 100%-tnu nukleotidnu i amnokiselinsku homolognost s golubljim cirkovirusom (pristupni broj AF252610.1) izdvojenim dokazanim u Njemačkoj. U jednog crvenokljunog labuda patološkohistološkom pretragom utvrđena je bolest odlaganja željeza (engl. iron storage disease /ISD/), dok su ostale ptice uginule pretežito od posljedica nastrijelnih rana i drugih vrsta traumatskih ozljeda (bijela roda, crvenokljuni labud, obični škanjac, orao štekavac, lastavica pokućarka, vrabac). Metodama dijagnostike molekularne biologije nisu izdvojeni virusi groznice Zapadnog Nila, ptičje gripeinfluence ptica i newcastleske bolesti.


Ključne riječi: slobodno živuće ptice, patološka morfologija, patološka histologija, lančana rekacija polimerazom (PCR), astrovirus, hepadnavirus, adenovirus, cirkovirus, koronavirus, galeb klaukavac (Larus cachinnans, Pallas 1811), divlja patka (Anas platyrhynchos), domaći golub (Columba livia domestica), crvenokljuni labud (Cygnus olor), bolest odlaganja željeza, amiloidoza



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