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Comparative clinico-pathological assessment of velogenic (sub-genotype

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Comparative clinico-pathological assessment ofvelogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens andpigeons

Aziz-ul-Rahman, Mohammed A. Rohaim, Rania F. El Naggar, GhulamMustafa, Umer Chaudhry & Muhammad Zubair Shabbir

To cite this article: Aziz-ul-Rahman, Mohammed A. Rohaim, Rania F. El Naggar, GhulamMustafa, Umer Chaudhry & Muhammad Zubair Shabbir (2019): Comparative clinico-pathologicalassessment of velogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian�avulavirus1 in chickens and pigeons, Avian Pathology, DOI: 10.1080/03079457.2019.1648751

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ORIGINAL ARTICLE

Comparative clinico-pathological assessment of velogenic (sub-genotype VIIi)and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens and pigeonsAziz-ul-Rahman a,g, Mohammed A. Rohaimb,c, Rania F. El Naggard, Ghulam Mustafae, Umer Chaudhryf andMuhammad Zubair Shabbir g

aDepartment of Microbiology, University of Veterinary and Animal Sciences, Lahore Pakistan; bDepartment of Virology, Faculty of VeterinaryMedicine, Cairo University, Giza, Egypt; cDivision of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University,Lancaster, UK; dDepartment of Virology, Faculty of Veterinary Medicine, University of Sadat City, Sadat, Egypt; eDepartment of Pathology,University of Veterinary and Animal Sciences, Lahore, Pakistan; fRoslin Institute, Easter Bush Veterinary Centre, University of Edinburgh,Roslin, Midlothian, UK; gQuality Operation Laboratory, University of Veterinary and Animal Sciences, Lahore Pakistan

ABSTRACTNewcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects a wide range ofavian species worldwide. Recently, several AAvVs of diverse genotypes have emerged withvarying genomic and residue substitutions, and subsequent clinical impact on susceptibleavian species. We assessed the clinico-pathological influence of two different AAvV 1pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feralpigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broilerchickens and pigeons. The velogenic strain caused 100% mortality in both avian specieswhile the mesogenic strain caused 0% and 30% mortality in chickens and pigeons,respectively. Both strains showed tissue tropism for multiple tissues including visceral organs;however, minor variances were observed according to host and pathotype. The observedgross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal andcloacal swabs, a comparable pattern of viral shedding was observed for both strains fromeach of the infected individuals of both avian species. The study concludes a varyingsusceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypesand, therefore, suggests continuous monitoring and surveillance of currently prevailingstrains for effective control of the disease worldwide, particularly in disease-endemic countries.

ARTICLE HISTORYReceived 26 March 2019Accepted 19 July 2019

KEYWORDSExperimental infection;currently prevailing strains;velogenic AAvV 1; mesogenicAAvV 1; chickens; pigeons

Introduction

Viral infectious diseases lead to serious economic lossesto the poultry trade worldwide (Haji-Abdolvahab et al.,2019). Among these, Newcastle disease (ND), causedby Newcastle disease virus (NDV) or Avian avulavirus1 (AAvV 1), is a highly contagious disease affectingmultiple avian species globally (Alexander & Senne,2008). The virus belongs to the genus Orthoavulaviruswithin the family Paramyxoviridae (Amarasingheet al., 2019). It has a mono-partite, negative-sensesingle-stranded RNA genome which is either 15186,15192 or 15198 nucleotides in length (Kolakofskyet al., 2005). The complete genome contains six codinggenes; nucleocapsid (N), phosphoprotein (P), matrix(M), fusion (F), haemagglutinin-neuraminidase (HN)and large polymerase (L) protein in the order 3′-NP-P/V/W-M-F-HN-L-5′ (Kolakofsky et al., 2005). Basedon pathogenicity to chickens, AAvV 1 strains are cate-gorized into four major pathotypes: velogenic (highvirulent), mesogenic (moderate virulent), lentogenic(low virulent) and asymptomatic (non-virulent)(Alexander et al., 2012). Subsequent to infection withvelogenic and mesogenic AAvV 1 strains, with a

mortality of 60-100%, infected birds exhibit clinicalsigns corresponding to the gastrointestinal, respiratoryand nervous systems depending upon the virus patho-type, target avian species and health status of birds(Kommers et al., 2001; Wakamatsu et al., 2006).

Despite mass vaccination and strict biosecuritymeasures, ND outbreaks are frequently observed inboth vaccinated and non-vaccinated flocks (Muniret al., 2012; Rehmani et al., 2015). AAvV 1 is a continu-ously evolving virus with evidence of accelerated evol-ution of the virulent strains (Fan et al., 2017). In thisregard, the isolation of novel avulaviruses of varyingpathogenicity and genotypes from a wide range ofhosts (Ramey et al., 2013; Shabbir et al., 2016; Brown& Bevins, 2017; Dodovski et al., 2017; Qiu et al.,2017; El Naggar et al., 2018; Aziz-ul-Rahman et al.,2018a, 2018b; 2019a, 2019b, 2019c) suggests a continu-ous virus evolution with subsequent implications fordisease spread (Solomon et al., 2012; Miller et al.,2015; Fan et al., 2017; Mayahi & Esmaelizad, 2017).Subsequent to evolution, the emergence of novel geno-types and sub-genotypes are now increasing over aperiod of time (Dimitrov et al., 2019a). The

© 2019 Houghton Trust Ltd

CONTACT Muhammad Zubair Shabbir shabbirmz@uvas.edu.pk Quality Operation Laboratory, University of Veterinary and Animal Sciences, Lahore54600, Pakistan

AVIAN PATHOLOGYhttps://doi.org/10.1080/03079457.2019.1648751

phylogenetic analysis revealed the circulation of 21genotypes globally and, among these, genotype VIand VII viruses are predominantly circulating andcausing several outbreaks in developing countriesincluding Pakistan (Liang et al., 2002; Sabra et al.,2017; Abolnik et al., 2018; Wajid et al., 2018; Ferreiraet al., 2019; Dimitrov et al., 2019b).

Owing to the distribution of various genotypes in aparticular region (Aziz-ul-Rahman et al., 2019c), theon-going outbreaks have been confirmed to be sub-genotypes of VI and VII viruses, which caused enor-mous mortality in susceptible hosts and also suggestedtheir panzootic potential (Miller et al., 2015). There-fore, it is imperative to perform a clinico-pathologicalevaluation of currently prevalent strains to determinehow the viruses are changing. The current study is anextension of our previous investigations of geneticcharacterization of Anseriformes- (Aziz-ul-Rahmanet al., 2018b) and pigeon- (Akhtar et al., 2016) origi-nated AAvV 1 strains because a full characterizationincluding genomic, genotypic and clinical pathogenesisof currently prevailing strains could also improvethe diagnostic aspects of disease and implementationof the control measures. Although the susceptibilityof chickens and pigeons to AAvV 1 infection hasbeen reported (Wakamatsu et al., 2006; Susta et al.,2011; Isidoro-Ayza et al., 2017), information relatedto comparative clinico-pathological assessment of cur-rently prevailing Anseriformes- and pigeon-originatedAAvV 1 viruses of varying pathogenicity and genotypesin chickens and pigeons is scarce. Besides continuousdisease monitoring, surveillance and subsequent gen-ome-based characterization, such substantial evidencecalls for experiments related to infectivity potential,transmission and shedding patterns of newly charac-terized isolates of varying pathogenicity in their suscep-tible hosts so that, if required, existing diseasesurveillance and control strategies could be revised indisease-endemic settings. Therefore, the current studywas intended to evaluate the infectious potential offield prevailing velogenic (sub-genotype VIIi) andmesogenic (sub-genotype VIm) strains in commercialchickens and pigeons under experimental and con-trolled conditions.

Materials and methods

Ethics statement

The study was carried out in accordance with therecommendations in the Guide for the Care andUse of Laboratory Animals by the National Institutesof Health and Animal Research Council (https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf). All protocols includingbird management, virus challenge, tissue and swabsampling, and sacrifice of birds were approved by the

Ethical Review Committee for Use of Laboratory Ani-mals (ERCULA) of University of Veterinary and Ani-mal Sciences (UVAS), Lahore (Permit Number:ORIC/DR-70).

Experimental challenge of velogenic andmesogenic AAvV 1 in chickens and pigeons

Two individual challenge studies were performed sep-arately for the assessment of the infectious potential ofpreviously characterized velogenic (MF437287; Anascarolinensis-II-UVAS-Pak-2015) (Aziz-ul-Rahmanet al., 2018b) and mesogenic (KU885949; Pigeon/MZS-UVAS-Pak/2014) (Akhtar et al., 2016) strainsin commercial broiler chickens and pigeons. The velo-genic strain was isolated from an asymptomatic duck(A. carolinensis) during a routine avian influenza sur-veillance programme, whereas; the mesogenic strainwas isolated from a ND outbreak in a feral pigeonflock. Recently genomic and genotypic analysis hascategorized the velogenic strain as a virus of sub-geno-type VIIi (Aziz-ul-Rahman et al., 2018b) while themesogenic strain was categorized as a virus of sub-gen-otype VIm (Akhtar et al., 2016). A total of 50 birds eachof clinically healthy broiler chicken (25 for velogenicchallenge experiment and 25 for mesogenic challengeexperiment) and pigeons (25 for velogenic challengeexperiment and 25 for mesogenic challenge exper-iment) were used. One-day-old broiler chicks wereprocured from an ISO certified commercial hatcheryand raised in the bird experiment unit at Quality Oper-ations Laboratory (QOL), UVAS, Lahore, Pakistan,until the beginning of experimentation. Healthypigeons were procured from the live bird marketlocated near Azadi Chowk (Minar-e-Pakistan), Lahore.Each challenge study was conducted for 39 days,during which chickens were housed in adequate facili-ties to attain maturity age till 27 days, while 2-month-old pigeons were housed for adaptation of captivity andvisual inspection of any clinical sign prior to challenge.Feed and water were provided ad libitum to all birdsalong with general bird care as recommended by theethical committee of the institute. All the birds werescreened for the existence of viral genome in orophar-yngeal and cloacal swabs, and antibodies in bloodagainst avian influenza virus (H9N2) and AAvV 1using reverse-transcriptase polymerase chain reaction(RT–PCR) and haemagglutination inhibition assay(HI) (Munir et al., 2012; Ali et al., 2017), respectively.Twenty-nine-day-old chickens and 62-day-old pigeonswere exposed to the selected virus pathotype. Theexperimental birds were randomly divided into sixgroups. For chickens, group A (n = 10) had birds thatwere challenged with the velogenic strain, group B (n= 10) represented contact birds while group C (n =05) served as mock or negative control. Similarly, forpigeons, group D (n = 10) had birds that were

2 AZIZ-UL-RAHMAN ET AL.

challenged with velogenic strain, group E (n = 10) rep-resented contact birds while group F (n = 5) served asmock or negative control (Figure 1). Regarding chal-lenge with velogenic AAvV 1 strain, groups A and Dwere exposed intranasally to 10−6.51 EID50/0.1 ml ofA. carolinensis-II-UVAS-Pak-2015. After 24 h of infec-tion, the contact chickens (group B) and pigeons(group E) were mixed together with challenged birds[group B within challenged chickens (group A) andgroup E within challenged pigeons (group D)], accord-ingly. In the second challenge experiment with meso-genic AAvV 1 strain, a similar approach wasemployed. For chickens, group A′ (n = 10) had birdsthat were challenged with the mesogenic strain,group B′ (n = 10) represented contact birds whilegroup C′ (n = 5) served as mock or negative control.Similarly, for pigeons, group D′ (n = 10) had birdsthat were challenged with mesogenic strain, group E′

(n = 10) represented contact birds, while group F′ (n= 5) served as mock or negative control. Regardingchallenge with mesogenic AAvV 1 strain, groups A′

and D′ were exposed intranasally to 10−6.87 EID50/0.1 ml of Pigeon/MZS-UVAS-Pak/2014. Mock (groupsC, C′ and F, F′) were inoculated with 0.2 ml phosphate-buffered saline (PBS). After 24 h of infection, the con-tact chickens (group B′) and pigeons (group E′) weremixed together with challenged birds [group B′ withinchallenged chickens (group A′)] and group E′ withinpigeons (group D′), accordingly. All birds were moni-tored daily for the clinical presentation of the diseasetill completion of the experiment.

Viral shedding, horizontal transmission andtissue distribution of AAvV 1

Oropharyngeal and cloacal swabs were collected asaseptically as possible from challenged (groups A, A′

and D, D′) and contact birds (groups B, B′ and E, E′)on 1, 3, 5, 7 and 9 days post infection (dpi), and pro-cessed for virus isolation and identification (Muniret al., 2012). For this purpose, all swab samples weretransferred into cryovials (2.0 ml) containing 1.5 mlbrain heart infusion medium with antibiotics (penicil-lin 2000 IU/ml, and gentamicin 200 μg/ml) and anti-fungal (fungizone 1.5 μg/ml). After centrifugation at2500 × g for 5 min, approximately 1.0 ml of eachsample was filtered through a 0.22 μm syringe filter(EMD Millipore Millex™, Millipore Billerica MA,USA). A 0.2 ml aliquot of the filtrate was inoculatedinto 9-day-old embryonated chicken eggs. The spothaemagglutination (HA) positive harvested allantoicfluid was tested for AAvV 1 using F gene-basedreverse-transcriptase polymerase chain reaction (RT–PCR) (Munir et al., 2012). For an assessment of tissuetropism, tissue samples (n = 19) including feather fol-licle, breast muscle, brain, whole eye, harderian glands,trachea, tongue, oesophagus, gizzard, proventriculus,

liver, heart, lungs, kidney, spleen, bursa, small intestine,caecum and caecal tonsils were collected from recentlydeceased/diseased birds showing typical signs of ND.These tissues were then processed individually forRNA extraction following the manufacturer’s instruc-tions (QIAmp Viral RNA extraction Mini Kit, Qiagen,Valenica, CA), and F gene-based molecular identifi-cation via RT–PCR (Munir et al., 2012).

Histopathological examination of tissues

In order to assess the severity of infection in chickensand pigeons to wild bird-originated velogenic andmesogenic AAvV 1 strains, the tissue samples were col-lected from birds immediately after death, and fromthose infected birds of groups A, A′, B, B′, D, D′, Eand E′, showing severe typical clinical signs of NDinfection (respiratory and/or nervous signs) regardlessof timeline. Selected tissue samples (brain, liver, tra-chea, lungs, kidney, spleen, bursa and intestine) werekept separately in 10% neutral buffered formalin(NBF) solution for haematoxylin and eosin stainingfor subsequent microscopic changes under a lightmicroscope (Carleton et al., 1980).

Results

Clinical presentation of velogenic AAvV 1infection in chickens and pigeons

The clinical presentation showed variations in severitylevel and duration of clinical signs between the chal-lenge (groups A and D) and contact birds (groups Band E) over time of post-infection. The challengedand contact chickens (groups A and B) showed 100%mortality by 6 dpi. In contrast, challenged and contactpigeons (groups D and E) showed 100% morbidity andmortality by 10 dpi. Briefly, the severity of clinical signswas more pronounced in chickens compared topigeons. The commonly observed clinical signs weregeneral sickness (anorexia, lethargy and depression),reluctance to move, respiratory distress (open mouthbreathing, sneezing, coughing, respiratory sounds, ocu-lonasal discharge) and diarrhoea as a gastric sign. Noneurological signs were noted in chickens (groups Aand B). Sudden death with no-to-negligible clinicalsigns was observed only in group A but no suddendeath was observed in any bird of groups D andE. Moreover, neurological signs were observed inpigeons of groups D and E. Birds of group A showedclinical signs from 2 dpi with mortality of three chick-ens. The clinical infection became exacerbated andpeaked at 3 dpi with the death of four chickens. After3 dpi, minor clinical signs were observed in one birdof group B. At the end of 4 dpi, the remaining chal-lenged chickens (group A) also succumbed. At 5 dpi,severe clinical presentation of ND was observed in

AVIAN PATHOLOGY 3

three birds of group B (contact chickens) and all con-tact birds died at the end of 6 dpi (Figure 2). Thepigeons of group D exhibited clinical signs from 3rddpi with mild clinical signs of respiratory distress.The clinical infection was exacerbated and peaked at4 dpi with the death of two pigeons in group D. At 5dpi, nervous signs were observed in a few birds ofgroup D with the death of three pigeons. After 5 dpi,mild clinical signs were observed in few birds ofgroup E. At the end of 6 dpi, two pigeons fromgroup D were found dead and clinical infection wasenhanced and peaked with death of three pigeons ingroup E. At the end of 7 dpi, two pigeons fromgroup D and four pigeons from group E were founddead. Subsequently, three deaths were observed inbirds of each group D and E at 8 dpi (Figure 2). Themock-infected (groups C and F) remained normalthroughout the experiment. Necropsy revealed charac-teristic lesions corresponding to ND infection andincluded haemorrhages in lungs and liver, enlargedliver, congested kidneys, mottled spleen, pinpointhaemorrhages in proventricular glands and oedema-tous bursa. The disease outcomes coupled with grosspathognomic lesions revealed a remarkable difference

in the susceptibility of the two avian species; chickens(groups A′, A, B, B′) and pigeons (groups D, D′, E, E′).

Virus shedding, horizontal transmission andvelogenic AAvV 1 tissue distribution

Throughout the whole experiment, the collected oro-pharyngeal and cloacal swabs showed a discrete patternof virus shedding without any noticeable differencewith respect to dpi within the same group of challengedchickens (group A) and contact chickens (group B).Virus shedding was detected in group A form 2 dpi.On the other hand, it was detected in group B from 4dpi. Similarly, for pigeons, virus shedding was detectedin pigeons (group D) from 3 dpi onwards while, in con-tact birds, it was detected from 5 dpi onwards. Mock-infected birds (groups C and F) remained virus-nega-tive throughout the whole experiment. In order toassess the tissue tropism of velogenic AAvV 1, the col-lected tissues (n = 19) from dead or diseased birds(pigeons and chickens) showing typical clinical signsand necropsy lesions were processed for detection ofthe viral RNA. For chickens (groups A and B), thevirus RNA was detected in all tissue samples. However,

Figure 1. Experimental layout for the assessment of infectious potential of velogenic and mesogenic AAvV 1 strains in chickens andpigeons.

4 AZIZ-UL-RAHMAN ET AL.

for pigeon (groups D and E), the existence of viral gen-ome was detected in all tissues except kidney, featherfollicle, heart and breast muscle.

Histopathological findings for velogenic AAvV 1infection

The velogenic AAvV 1 strain caused notable histo-pathological lesions in morbid birds of both avianspecies. For chickens, the microscopic findings wereconsistent with the afore-mentioned gross lesionssuch as congestion, haemorrhages with mononuclearinflammatory cell infiltration in sub-mucosa of trachea,lung, kidney and spleen, a mild congestion in brain,degeneration of hepatocytes, fatty changes (vacuolesin hepatocytes), infiltration of inflammatory cells inthe portal cord of the liver, damaged basal membraneand degeneration in follicles in bursa of Fabriciusalong with presence of dead/necrotic tissue masses,and sloughing of epithelium in the small intestine. Incontrast, the microscopic findings were comparativelyless pronounced in pigeons (groups D and E) than tochickens (groups A and B). Lesions included mild con-gestion in brain, disruption of cardiac fibres withaccumulation of immune cells in heart, congestion inparabronchial blood capillaries, haemorrhages withmononuclear inflammatory cells infiltration in lungs,fatty changes evidenced by vacuoles of varying sizes

in the cytoplasm of hepatocytes, infiltration of inflam-matory cells in the portal cord, engorgement of sinusoi-dal capillaries with red blood cells and Kupffer cells inliver, sloughing of epithelium in the small intestine,and infiltration of inflammatory cells in the gizzard(Figure 3).

Clinical presentation of mesogenic AAvV 1infection in chickens and pigeons

Based on severity and duration of clinical infection,variations in the clinical signs were observed amongchallenged birds (groups A′ and D′) and contactbirds (groups B′ and E′) over a designated time period.The challenged and contact chickens (groups A′ andB′) showed 40% morbidity without any mortality by10 dpi. Notably, neurological signs were also observedin chickens of groups A′ and B′, however, no suddendeath was observed in any group. Birds of group Ashowed mild clinical signs from 5 dpi onward. Minorclinical signs were observed in the birds of group B′

by 6 dpi (Figure 4). The challenged and contact pigeons(groups D′ and E′) showed 50% morbidity and 30%mortality by 10th dpi. Clinical signs, including respir-atory and nervous signs, were also observed in pigeonsof groups D′ and E′. Similar, but less severe, clinicalsigns were observed in all birds as reported in the pre-vious velogenic-challenge experiment. The birds of

Figure 2. Percentage survival rates of chickens (groups A, B and C) and pigeons (groups D, E and F) following infection with velo-genic AAvV 1 strain compared with control birds.

AVIAN PATHOLOGY 5

group D′ showed clinical signs from 6 dpi. At 7 dpi,mild nervous signs were observed in a few birds ofgroup E. After 8 dpi, mild respiratory signs were alsoobserved in few birds of groups D′ and E′ (Figure 4).The control birds (groups C′ and F′) remained normalduring the whole experiment. Upon post mortem

examination of infected/morbid birds, characteristicnecropsy lesions, including haemorrhages and conges-tion in brains and lungs, enlarged liver, congested kid-neys, mottled spleen, pinpoint haemorrhages inproventricular glands and oedematous bursa, wereobserved.

Figure 3. Microscopic examination of histopathologic changes in different tissues collected from pigeons infected with velogenicstrain. Arrows indicate histologic and pathologic lesions in (A) lung, (B) brain, (C) liver, (D) small intestine, (E) gizzard and (F) heart.

Figure 4. Percentage survival rates of chickens (groups A′, B′ and C′) and pigeons (groups D′, E′ and F′) infected with mesogenicAAvV 1 strain compared with control birds.

6 AZIZ-UL-RAHMAN ET AL.

Virus shedding, horizontal transmission andmesogenic AAvV 1 tissue distribution

Virus shedding was detectable in challenged chickensof group A′ from 5 dpi onward, whereas the viruswas detected in swabs of contact chickens of group B′

from 6 dpi onward. Throughout the whole experiment,the oropharyngeal and cloacal swabs showed a discretepattern of virus shedding without any noticeable differ-ence with respect to dpi within the same group of chal-lenged and contact chickens (groups A′ and B′).Similarly, for pigeons, virus shedding was detectablein challenged pigeons (group D′) from 6 dpi onwardwhile, in contact pigeons (group E′), it was detectedfrom 7 dpi onward. Collectively, the virus was detectedin cloacal swabs from 6 dpi onward, while it was detect-able from 7 dpi onward in oropharyngeal swabs. Mockbirds (groups C′ and F′) remained virus-negativeduring the whole experiment.

In order to assess the tissue tropism of mesogenicAAvV 1, the tissues (n = 19) collected from birds(pigeons and chickens) showing typical clinical signsand necropsy lesions were processed for the detectionof viral RNA. For chickens (groups A′ and B′), thevirus RNA was detected in most of the tissue samplesexcept heart, kidney, feather follicle, tongue, oesopha-gus, caecum, caecal tonsils and liver. However, forpigeons (groups D′ and E′), the presence of viral gen-ome was detected in all tissues except heart, kidney,breast muscle, feather follicle, tongue and bursa ofFabricius.

Histopathological findings for mesogenic AAvV1 infection

The histopathological changes, including haemor-rhages with mononuclear inflammatory cell infiltrationin some areas of tracheal tissues coupled with absence

of pseudostratified columnar epithelium in trachea,haemorrhages and congestion with degenerativechanges in the lamina propria of lung, congestion inperitubular capillaries, cellular swelling in renal epi-thelial cells and coagulative necrosis in renal tubuleswere observed in tissues collected from morbid chick-ens. Some renal tubules had epithelial cells separatedfrom the basement membrane, congestion, haemor-rhages and necrotic degeneration in spleen, mild con-gestion in brain, venous congestion, degeneration inhepatocyte, fatty changes (vacuoles in hepatocytes),infiltration of inflammatory cells in the portal cord inliver, damaged basal membrane and degeneration infollicles in bursa of Fabricius and presence of dead/necrotic tissue mass, sloughing of epithelial cells inthe lumen, dropout of intestinal villi in the small intes-tine (Figure 5).

In contrast to chickens, the histopathological altera-tions were comparatively less pronounced in pigeons(groups D′ and E′). Microscopic lesions included con-gestion, haemorrhages with mononuclear inflamma-tory cells infiltration in lung, mild congestion inbrain, fatty changes evidenced, infiltration of inflam-matory cells in portal cord of liver, presence of dead/necrotic tissue masses, dropout of epithelium in smallintestine, infiltration of inflammatory cells in gizzardand disruption of cardiac fibres in heart. None of thetissues collected from birds of the control group hadany apparent histological and pathological changes(Figure 6).

Discussion

The experimental studies under controlled conditionsenable us to properly evaluate the infectivity and trans-missibility of field prevailing AAvV 1 strains, includingonset of clinical signs, mortality, virus shedding andtransmission to healthy birds (Panshin et al., 2002;

Figure 5. Microscopic examination of histopathologic changes in different tissues collected from chickens infected with the meso-genic strain. Arrows indicate histologic and pathologic lesions in (A) trachea, (B) lung, (C) kidney, (D) spleen, (E) brain, (F) liver, (G)bursa of Fabricius and (H) small intestine.

AVIAN PATHOLOGY 7

Kapczynski et al., 2006; Wakamatsu et al., 2006; Car-rasco et al., 2008; Alexander et al., 2012; Guo et al.,2014; Desingu et al., 2017). Therefore, owing to thegreater prevalence of genetically diverged isolatesthan previously known, the current study was designedto evaluate the comparative pathogenicity of currentlycirculating AAvV 1 strains of varying pathogenicity(velogenic and mesogenic strains) in commercialchickens and pigeons. The oculonasal route was usedto induce the infection in both avian species simplybecause it is considered a natural route of infection infield conditions (Alexander et al., 2012). In fact, withthe same route of infection, true clinical presentationof ND has been evidenced in various susceptibleavian species in captivity (Otim et al., 2006; Piacentiet al., 2006; Carrasco et al., 2008; Susta et al., 2010;Guo et al., 2014; Kang et al., 2016: Desingu et al.,2017). We found a higher rate of morbidity and mor-tality in both avian species upon exposure to a wildbird-originated velogenic AAvV 1 strain than uponexposure to a pigeon-originated mesogenic AAvV 1strain. Similarly, velogenic strains have been frequentlyreported from chickens and pigeons with high morbid-ity and mortality (Aldous et al., 2010; Alexander et al.,2012), whereas mesogenic strains showed relatively lessmortality in both commercial chickens and pigeons(Kommers et al., 2001; Kim et al., 2008). In fact, velo-genic strains caused a high degree of infection in chick-ens, while; mesogenic strains show a high degree ofinfection in pigeons (Kommers et al., 2001; Kimet al., 2008). This is because; the course of infectionwith a pigeon-originated AAvV 1 variant in chickenscan be very mild even though the strain is mesogenicdue to its classification into pathotypes, which is actu-ally dependent on virulence assessment via intracereb-ral inoculation in one-day-old chickens but not in

pigeons (Meulemans et al., 2002). Along with intracer-ebral pathogenicity index in one-day-old chickens, dis-ease severity in susceptible hosts could serve as areference index for AAvV pathotyping. However, theaccuracy and precision of these indexes are questionedto some extent, especially when used to evaluate virusesisolated from wild birds (Guo et al., 2014).

There is still controversy over the susceptibility ofchickens and pigeons to viruses of varying pathogen-icity. For instance, a few studies demonstrated a diseaseof less severity in chickens upon challenge with virusesof velogenic and mesogenic strains (Panshin et al.,2002; Guo et al., 2014). A severe form of infectionwas observed in chickens upon challenge with virusesof velogenic and mesogenic strains (Ezema et al.,2016; Ren et al., 2017; Xue et al., 2017). Likewise, a vari-able degree of infection was also observed in pigeonsinoculated with velogenic and mesogenic strains(Wakamatsu et al., 2006; Guo et al., 2014; Carrascoet al., 2016). However, some of the studies arguedthat wild bird- (including pigeon) originated avula-viruses need further passages in embryonated chickeneggs in order to adapt and subsequently infect chickens(Kommers et al., 2003). Mesogenic strains of pigeonorigin may evolve into virulent viruses and, therefore,lead to major outbreaks by causing infection in chick-ens (Dortmans et al., 2011a). Differences in the suscep-tibility of chickens to wild bird-originated AAvV 1strains were observed because wild bird originatedstrains may gain virulence after a variable number ofpassages which was not performed in the currentstudy. Therefore, host specificity is not only a typicalcharacteristic of wild bird-originated AAvV 1 strainsbecause a vast range of hosts is susceptible to differentstrains. Such findings, coupled with the observationreported in this study, highlight the potential of wild

Figure 6. Microscopic examination of histopathologic changes in different tissues collected from pigeons infected with the meso-genic strain. Arrows indicate histologic and pathologic lesions in (A) lung, (B) brain, (C) liver, (D) small intestine, (E) gizzard and (F)heart.

8 AZIZ-UL-RAHMAN ET AL.

bird-originated strains to cause infection in commer-cial chickens (Karamendin et al., 2016).

The clinico-pathological findings of the currentstudy revealed nervous signs in both avian speciesupon challenge with mesogenic strains, whereas ner-vous signs were observed exclusively in pigeons whenchallenged with the velogenic strain. Similar to findingsreported previously (Kapczynski et al., 2006; Waka-matsu et al., 2006; Carrasco et al., 2008), this indicatesa varying pattern of tissue tropism of both species foreach of the avulaviruses and the fact that chickens andpigeons are not equally susceptible to infection by thesame strain (Wakamatsu et al., 2006). Indeed, variationsin disease severity involving varying tissues have beenwell documented amongmultiple avian species exposedwith the same strain of avulavirus (Piacenti et al., 2006;Kang et al., 2016). A development of viscerotropic andneurotropic form of infection has been reported pre-viously when chickens were inoculated with the velo-genic strain (Oladele et al., 2005; Bobbo et al., 2013;Igwe et al., 2014), whereas only the neurotropic formof infection was evidenced in pigeons upon exposureto the velogenic strain (Kapczynski et al., 2006). Thevelogenic strain, isolated from an epidemic in a flockshowing both respiratory and nervous signs, exhibitedonly a neurotropic form of infection following exper-imental infection in chickens (Igwe et al., 2014). Invol-ving the central nervous system and respiratory tract,mild to moderate lesions in chickens have previouslybeen reported upon exposure to a mesogenic strain(Wakamatsu et al., 2006; Susta et al., 2010). A possiblereason for such variations in tissue tropism, and a sub-sequent variable degree of infection in multiple avianspecies, could be linked to the presence of proteases orfurin-like enzymes in different tissues of susceptible/infected hosts (Seal et al., 2005).

Consistent with previous observations (Panshinet al., 2002; Desingu et al., 2017), when challengedwith velogenic and mesogenic strains, we evidenced avariable pattern of viral shedding from both avianspecies. Viral shedding was detected in both orophar-yngeal and cloacal swabs in a pattern comparable toprevious studies (Kapczynski et al., 2006; Wakamatsuet al., 2006) where the virus was detected from oraland tracheal swabs starting from 2 dpi. We founddetection of virus in cloacal swabs earlier than orophar-yngeal swabs. Some minor variations, in this regard,have previously been documented. For instance, simi-lar to our study observations, the onset of virus shed-ding was detected earlier in cloacal swabs startingfrom 2 to 3 dpi (Kapczynski et al., 2006; Wakamatsuet al., 2006; Carrasco et al., 2009). Likewise, in a pre-vious study, virus shedding through the cloaca wasalso observed from 5 dpi onward (Carrasco et al.,2008). In contrast, the onset of virus shedding wasdetected in oropharyngeal swabs starting from 5 to 9dpi (Dortmans et al., 2011b; Dai et al., 2014). The

NDV infection was also evident in contact chickensand pigeons where virus distribution among differentorgans or tissues was identified. Such type of trans-mission is not uncommon for velogenic or mesogenicAAvV 1 strains (Shabbir et al., 2016; Desingu et al.,2017). Similar to previous studies, a variable patternof tissue tropism was evidenced depending on thepathotypes used for challenge and the avian speciesinvolved (Pandarangga et al., 2016; Shabbir et al.,2016; Igwe et al., 2018). Though comparable, in con-trast to chickens, the distribution of viral RNA wasnot detected in all of the tissue samples collectedfrom pigeon. Such a pattern of varying tissue tropismhas also been evidenced previously (Guo et al., 2014).Taken together, the current study revealed that bothtypes of AAvV 1 strains have the potential to targetmultiple tissues which, therefore, could be utilized foreither detection and/or isolation of the virus. In thisregard, the potential of molecular assays such as RT–PCR has previously been validated for accurate detec-tion of the viral genome using a range of tissues fromthe diseased/deceased host (Barbezange & Jestin, 2002).

Conclusion

Although there are variations in disease severity, trans-mission, virus shedding, and tissue tropism, wild bird-originated velogenic and mesogenic AAvV 1 strainshave the potential to induce infection in both chickensand pigeons. These wild bird-originated AAvV 1strains are circulating in the environment, may causeinfection when coming into contact with commercialbirds, and outbreaks may occur, with considerableeconomic losses to poultry industries particularly indisease-endemic countries. A lack of appropriate biose-curity measures is not uncommon in developingcountries where disease is endemic and disease occur-rence is frequent. Therefore, integrated continuous dis-ease surveillance, coupled with biosecurity measuresincluding vaccination regimen of available live andkilled ND vaccines, are crucial for an efficient diseasecombat in a disease-endemic setting.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

Aziz-ul-Rahman http://orcid.org/0000-0002-3342-4462Muhammad Zubair Shabbir http://orcid.org/0000-0002-3562-007X

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