Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during in India

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1 Indian JoumalOf Experimental Biology Vol. 41, Aprif2003, pp Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during in India Samadhan J Jadhao b, J N Deepak " J M Kataria", R S Kataria', A K Tiwari', R Somvanshi", P Sangamithra b & K C Verma' "Division of Avian Diseases, Indian Veterinary Research Institute, (ICAR), Izatnagar, , India bhigh Security Animal Disease Laboratory, Indian Veterinary Research Institute Campus, (lcar), Anand Nagar, Bhopal, , India Received 20 June 2002; revised 19 February 2003 In the present study characterisation has been done for six group I fowl adenoviruses (FA V) isolated from outbreaks of infectious hydropericardium (IHP) of chickens that occurred in different states/regions of India during the years These six viruses were identified as FAV serotype 4 by virus neutralisation and restriction endonuclease analyses. Antigenic analyses of the viruses revealed close relationship (R-values ). Under the experimental conditions, we have been able to induce IHP using FA V serotype 4 isolate AD: 411 and were also able detect FA V antigens in myocardial tissues by immunofluorescence assay (a new observation), an indication that IHP causing FA V serotype 4 strain replicate in myocar-. dial tissue. Restriction endonuclease analysis of the viral genomes (approximately 46 Kb), using Hind III, Sma I, Xba I, Bam HI, Pst I and Dra I produced identical genetic profiles. Pst I and Bam HI profiles for these six vitus isolates were identical to those published earlier for an IHP causing Pakistani FA V serotype 4 isolate KR31. The identical genetic profiles of viruses, chronology of the outbreaks of IHP in Pakistan during 1989 onward and later in Jammu and Kashmir, India (1994), suggest that FA V serotype 4 isolates involved in outbreaks of IHP in India had probably spread from Pakistan. In order to prevent further spread and economic losses due to IHP in India, based on the antigenic relatedness data in this paper, anyone of the six studied FAV serotype 4 isolates can be used as a candidate for mass production of CEH culture based killed vaccine. A new disease of chickens called hydropericardium syndrome was reported during 1989 in Angara Goth of Pakistan 3. Use of term 'infectious hydropericardium' (IHP) was suggested instead of hydropericardium syndrome because of infectious nature of FA V serotype 4 used to reproduce the hydropericardium in young chickens l2. IHP is characterised by pulmonary edema, discoloured friable liver, nephritis and accumulation of straw coloured or jelly like fluid in pericardial sac and heavy mortality in young broilers. An adenovirus was detected in the liver of affected birds by electron microscopy6. Later on, in early 1994, IHP was reported in Jammu and Kashmir, India 9. This disease has rapidly spread to different parts of the country leading to heavy mortality (60-75%) in the affected broiler flocks II. During the same period, several outbreaks of IHP caused by FA V serotype 4 had been reported from South American countries, viz. Peru, Ecuador 23, Russia and JapanlS. So far 12 serotypes are recognised within the group I fowl adenoviruses by cross neutralisation assay I. The major antigenic protein of adenoviruses is hex on on which type; group and subgroup antigenic deter- *Correspondent author : jmohan@irri.up.nic.in minants are located l9. The group specific antigenic determinants are common to all 12 FA V serotypes, whereas type specific antigenic determinants are restricted to a particular serotype. Some of the FA V isolates clearly fit into one serotype but some crossreact with other serotypes and are difficult to classify I4.16. Comparison of virus isolates using polyclonal antiserum is not necessarily a representative of the viral genomes. Besides serological typing, restriction endonuclease (RE) analysis of fowl adenoviral genome has been recommended as an adjunct rapid molecular tool for typing ' and discrimination of strains within a type5.s Because of the occurrence of multiple serotypes (FA V 1-12) in group I fowl adenovirus, birds vaccinated with one serotype are not protected to the virulent challenge with other serotype(s)14. Since 1996, IHP has spread widely in Indial! and the viruses involved in various outbreaks of IHP had not been characterized antigenically and genetically except our earlier report on serological identification of involvement of FA V serotype 4 in IHPII. The control of IHP on various broiler chicken farms of India 21 and Pakistan 2 is being done by the use of inactivated vaccine prepared from liver homogenates of IHP affected chickens. In view of developing an

2 322 INDIAN J EXP BIOL, APRIL 2003 effective vaccine and control strategy, the aim of this work was to characterize FA Vs isolated from six different field outbreaks of IHP, that occurred during in various states/regions of the country and to understand the epidemiology. In addition we have examined the pathogenicity of one of these isolates by oro-nasal infection of 2-week-old FA V antibody free broiler chickens. Materials and Methods Clinical specimens - Liver tissues were collected aseptically (in 50% buffered glycerol) from the broiler chickens died in outbreaks of IHP in various organized broiler farms of different states/regions of India during (Table 1). Agar gel immunodiffusion assay-twenty per cent (w/v) liver homogenates in PBS were treated with 50% (v/v) reagent grade chloroform to disrupt the liver cells. The homogenates were centrifuged at 6000 rpm for 20 min and aqueous supernatants were tested in agar gel immunodiffusion assay (AGIO) against rabbit anti FA V serotype 1 (CELO) hyper immune serum". Chicken embryos and chickens-twelve day old embryonated chicken eggs and unvaccinated one-dayold chicks were procured from experimental hatchery unit of Central Avian Research Institute, Izatnagar. The chicks were raised up to 14 days in the experimental house. Before starting of the experiment, birds were tested for presence of antibodies to avian reovirus, Newcasile disease virus (NDV) and infectious bursal disease virus (lbdv) by serum neutralisation assay, and group I adenovirus by., AGID assay using FA V serotype 1 (CELO) antigen'. Virus isolation - The primary chicken embryo hepatocytes (CEH) cultures were prepared from 13 day old chicken embryos'. The samples that were found positive for group I FA V antigens in AGIO assay were used as inoculums to isolate virus. Virus isolation was performed in CEH cultures up to four passages. The median infectivity titers of the viruses were determined in CEH microtitre cultures 20 The viruses were plaque purified (agarose overlay) once using confluent CEH cultures. The single plaque from each virus isolate was amplified in CEH cultures for further work. Immune antiserum - Immune homologous antiserum was raised against each of the six virus isolates (plaque purified) in a group of eight FA V antibody free chickens (1 week old). Each bird was inoculated intramuscularly with a single dose of (100 TCIDsolO.5 ml) FA V serotype 4 virus isolate. The inoculated chickens were kept under isolation and blood was collected 30 days post infection (d.p.i.) by puncture of wing vein for separation of serum. Serotyping and antigenic relationship - Cross neutralisation test for FA V serotyping was performed using 100 TCIDsoI50 Jll of virus in microneutralisation assai. Dr J B McFerran, Veterinary Research Laboratory, Stormount, Belfast, Northern Ireland, provided the standard rabbit FA V typing sera against FA V serotypes Antigenic relationship between the virus isolates was estimated by neutralisation assay using immune antiserum to each virus. The constant serum (l: 10 dilution) and log dilutions of the virus were used in the test. The infectivity titers of the viruses in the absence and presence of homologous and heterologous immune antiserum were determined 20, neutralisation indices were calculated and antigenic relationship was estimated 4 Viral DNA extraction and restriction endonuclease analysis-the virus infected CEH cultures were harvested at 72 hr post infection at 70-80% CPE. The viral DNA was extracted from the purified virus s. Table I - Details of FA V serotype 4 isolates used in the present study Virus isolate Year Origin from India Details AD : Gurgaon Commercial broilers, age 15 days, 60% mortality, AD : Barcilly Commercial broilers, age 20 days, 65% moitality, AD : 3!!!996 Pune Commercial broilers, age 11 days, 73% mortality, AD: Bangalore Commercial broilers, age 17 days, 69% mortality, AD: Hyderabad Commercial broilers, age 26 days, 67% mortality, AD: Coimbatore Commercial broilers, age 20 days, 75 % mortality,

3 JADHAO et at.: CHARACTERISATION OF FOWL ADENOVIRUSES FROM CHICKENS 323 Restriction endonuclease (RE) cleavage analysis of viral DNA was carried out using Pst I, Dra I, Xba I, Sma I, Hind III and Bam HI enzymes as recommended by the manufacturer (Promega Inc., USA). Restriction fragments were analysed by electrophoresis in agarose gel (0.7%) containing ethidium bromide (0.5 Ilg/ml), photographed on UV transilluminator and molecular sizes of DNA fragments were determined on a standard curve prepared by use of molecular size using a semilogarithimic paper. Pathogenicity evaluation - Fifty broiler chickens at the age of 14 days were inoculated with a dose of 10 3 TCID mi, FAV 4 AD: 411 virus via oronasal route. Ten chicks were given PBS and kept as controls. All the chickens were examined daily during the experimental period of 21 days. Birds that died were necropised, and tissues from liver, heart, kidney, caecal tonsils, spleen, thymus and bursa of Fabricius were sampled aseptically and frozen at -20 C for detection of FA V antigens by immunofluorescence assay (IFA) and recovery of virus. Tissues from these organs were also fixed in 10% neutral buffered formalin for histopathology. The birds that recovered following oro-nasal infection were bled to collect sera on 21 days post infection (dpi). The recovered birds were killed by ether inhalation and subjected for necropsy. The tissues were also collected from different organs and used for FAT, virus recovery and histopathology. FLuorescent antibody assay and recovery of virus Cryosections of liver, heart, kidney, caecal tonsils, spleen, thymus and bursa of Fabricills were treated with 1:50 dilution of chicken anti-fay serotype 4 (AD: 411) serum. The antigen and antibody complex in the tissue sections were traced with rabbit antichicken IgG labelled with FITC (Promega, USA), and evaluated under fluorescent microscope. The selected tissues of liver, heart, kidney, caecal tonsils, spleen, thymus and bursa of Fabricius collected from the birds died during pathogenicity trial or sacrificed at the end of the experiment, were processed for virus isolation as described earlier. The recovered viruses were again subjected for type identification using standard antiserum against 12 different FA V serotypes. Results Virus isolation-the liver homogenates gave a single precipitation line with hyper immune serum against FA V serotype 1 (CELO). Virus isolates AD: 157, AD: 411, AD: 317 and AD: 421 produced CPE characterised by refractivity and ballooning of the cells and detachment of the cultures from surface of the flask in the form of bunches of grapes on first passage. The isolates AD: 185 and AD: 311 produced similar changes in second passage. The virus isolates achieved infectivity titer between to TCID501ml. Serotyping and antigenic relationship-ail the six virus isolates were neutralised effectively by the reference antiserum to FAV serotype 4 strain KR5 (reciprocal serum neutralisation titer of 3200) and FA V serotype 11 strain C2B (reciprocal serum Table 3-Calculated sizes (Kb) and pattern of restriction fragmenls of viral genomes of the FA V serotype 4 isolates Restriction enzymes Fragment Pst I Dra I Bam HI A B C D E F G H I J 0.20 Total *Molecular sizes of DNA fragments generated by digestion of viral DNA with Hind III are not shown as the total size of the three fragment generated was less than expected, probably because of generation of several small fragments run out in 0.7% agarose gels. Table 2 - * Antigenic relationship (R) among the FA V serotype 4 isolates Immune antiserum Virus AD: 157 AD: 185 AD:311 AD: 317 AD: 411 AD:421 AD: AD: AD: AD: AD: AD: * Antigenic relationship (R") =" r1.r2 (Archetti and Horstfall, 1950).

4 324 INDIAN J EXP BIOL, APRIL 2003 Table 4-Persistence of adenovirus in various tissues of chickens infected orally with FA V serotype 4 isolate AD: 411 Days No. of FA V Antigen detection b~ FAT Virus isolation in CEH cultures P.I. chicks L S B T CT K H L S B T CT K H 5 t n= ND 6 t n= ND 7t n= ND 8 t n= ND 9t n= ND 10* n= ND 11* n= ND 12* n= ND 13* n=3 + + ND 14* n=3 + + ND 15* n=3 ND 16* n=3 ND Control n=5 ND Days P.I-days post infection, L- liver, S- spleen, B- bursa, T- thymus, CT- cecal tonsils, K- kidney, H- heart (myocardium); n - number of chicks examined; t - chickens died after infection with FA V serotype 4; * - chicks sacrificed periodically; (+) - positive;-- negative; and ND- not done I 1 J I~., (, 7 8 M <) I 2 J M 4 56 M 7 8<) MI2.>,'t')6.. Hind III Pst I. Ora I Hind III Pst I Dra I Bam HI A 8 c Fig. I - Digestion patterns of fowl adenovirus serotype 4 isolate DNA generated by different restriction endonuclease (A) - Hind Ill, Pst I and Dra I. [Lanes 1,5 and 9 = AD: 157; lanes 2,6 and IO = AD: 185; lanes 3,7 and 11 = AD: 311; lanes 4,8 and 12 = AD: 317; M= lambda-phage DNA digested by Hind III and Eco RI); (B)-Hind III, Pst I and Dra I. [Lanes 1,4 and 7 =AD: 317; lanes 2,5 and 8=AD: 418; lanes 3,6 and 9=AD: 421; M= lambda-phage DNA digested by Hind III and Eco Rl); and (C)-Bam I. [Lane 1 =AD: 157; lane 2 =AD: 185; lane 3 =AD: 311; lane 4= AD: 317; lane 5= AD: 418; lane 6= AD: 421; M= lambda-phage DNA digested by Hind III and Eco RI)

5 JADHAO et al.: CHARACTERISATION OF FOWL ADENOVIRUSES FROM CHICKENS 325 neutralisation titer of 800). The reference antisera other than KR5 and C2B did not neutralise the virus isolates (serum neutralisation titer <50). The antigenic relationship (R values) among the FAV serotype 4 isolates is shown in Table 2. Restriction endonuclease (RE) analysis-re profiles for each of the six FA V isolates were identical with any of the six REs used. The molecular size of the viral genome was approximately 46 Kb (Table 3). The representative RE patterns of the viral DNA with enzymes Hind III, Pst I, Dra I and Bam HI are shown in Fig. la, B, C. Pathogenicity evaluation - The infected birds showed clinical signs between 3 to 5 dpi and were characterised by anorexia, depression, ruffled feathers, closed eyes and loose droppings. Fourty two per cent (21/50) mortality occurred between 5 to 9 dpi. The remaining sick chickens recovered from the clinical signs by 12 dpi and appeared healthy between 13 to 15 dpi. The necropsy of the chickens that died during the experimental period showed enlarged livers with multiple pinpoint hemorrhages on the surface, hydropericardium and patechial hemorrhages on the epicardium, congested kidneys and caecal tonsils. The spleen, thymus and bursa of Fabricius showed significant (p<0.0l) atrophy (data not shown) and the thymic lobes showed pinpoint hemorrhages. On histological examination, the liver showed fatty degeneration in parenchyma, infiltration of lymphocytes, focal necrosis, large basophillic intranuclear inclusion bodies in many hepatocytes. The changes in the heart were focal or multi focal hemorrhages along the myofibrils, edema in pericardial and myocardial regions. Spleen, bursa of Fabricuous, thymus and caecal tonsils showed lymphoid depletion. Fluorescent antibody assay and recovery of virus The FA V antigens could be detected by indirect immuno-fluorescence assay in liver, caecal tonsils, myocardial tissue, bursa of Fabricius, thymus and spleen but not in kidneys of all the chickens that died following inoculation of the virus. The serotype 4 FA V could be recovered from all those organs that were positive for presence of FA V antigens as examined by IFA. Discussion Since the first report of an adenovirus infection in chickens 25, FA Vs have been found to be associated with a variety of diseases of chickens. Of the three groups of avian adenoviruses, the conventional group I adenoviruses are reported to cause a new disease of chickens known as infectious hydropericardium or hydropericardium syndrome 3, 7,12,17. In the present study, we investigated outbreaks of IHP that occurred in different parts of the country during to gain information about the prevalence of FA V serotypes, antigenic relationship, genome types, epidemiological information and pathogenic potential of a virus isolate to the chickens under experimental condition. The liver tissue of the birds obtained from field outbreaks of IHP reacted positively in AGIO with antiserum to FA V serotype 1 (CELO) indicating that the viruses belonged to fowl adenovirus group I. This reactivity occurs because all the 12 serotype of FAV share a common group specific antigen 13. Infection of CEH cultures using the AGIO positive liver homogenate induced CPE characteristic of adenovirus l4. Cross neutralisation of the virus isolates with a panel of reference FA V serotype 1-12 antisera revealed that the viruses belonged to a single serotype i.e., FA V 4. The adenoviruses isolated from outbreaks of IHP in Asia (Pakistan and Japan), Europe and South American countries have been identified as FA V serotype 4. All the six isolates of FAV serotype 4 interestingly exhibited partial cross neutralisation with antiserum to FAV serotype 11 strain C2B. In earlier molecular or and serological studies, it has been suggested that FA V serotype 4 strain KR5 and FA V serotype 11 strain C2B should be considered as into one serotype 8. Similar observations have been recorded lo. 24 earlier for FA V serotype 4 and FA V serotype 11. Since the antigenic relationship (R-values) for the six FA V serotype 4 isolates are in the range of 0.93 to 0.96, they were antigenic ally closely related. The genetic profiles of these six FA V serotype 4 strains were identical as analysed by digesting DNA with restriction enzymes. It was not possible for us to include in our study, the prototype FAV serotype 4 strain KR5 and Pakistani FA V serotype 4 strain KR31 due to restriction on import of infectious viruses for biosafety reasons. The published 12 Bam HI and Pst I restriction profiles for Pakistani FA V serotype 4 strain KR31 are identical to the six virus isolates studied by us. However, the RE profiles of the viruses used in this study are similar but not identical to the published RE profiles of standard FAV serotype 4 strain KR5. The isolates could be classified as members of DNA group C based on Hind III/Bam HI restriction classification criteria established for genome typing of group I fowl adenoviruses 26 The genome size of each of the FA V serotype 4 isolates was approximately 46 Kb, which is in agreements with FA V serotype 4 strain CFA 15.

6 326 INDIAN J EXP SIOL, APRIL 2003 Pathogenicity test conducted by infection of twoweek-old broiler chickens via natural (oro-nasal) route resulted in 42% mortality with hydropericardium as a consistent necropsy finding in all dead chickens indicating that isolate AD: 411 indeed was highly pathogenic and able to induce hydropericardium experimentally. In this study, as per the history of IHP outbreaks, there was mortality between 60 to 75% under field situation, but under the experimental condition the virus isolate AD: 411 caused 42% mortality, which was lower compared to field situation. Under the field situations the role of other viruses/pathogens and their contributory effects in causation of increased mortality rates could not be excluded and needs further investigation. No antibody response could be detected in the infected and control chickens against IBDV, NOV and avian reovirus indicating that the birds were not exposed to any of these viruses during experiment. We could detect the group I adenoviral antigens in the tissues of heart, liver, caecal tonsils, bursa of Fabricius, spleen and thymus of the oro-nasally inoculated birds that died 5 to 9 dpi FA V serotype 4 could be recovered in CEH cultures from organs that were positive by FAT. This observation indicated that FAT was as sensitive as virus isolation. We examined heart tissues by FAT because of the consistent hemorrhages on epicardium of heart. To our knowledge there is no report on detection of FA V antigen in myocardial tissue of the bird died of either experimental or natural IHP. This finding indicated that IHP causing FAV serotype 4 replicated in myocardial tissues and the fluid, which accumulated in pericardial sac, might be infectious. The Pakistani FA V serotype 4 strain KR 31 responsible for IHP is reported to persist in liver and caecal tonsil of experimentally infected chickens upto 11 dpi onlyl2. The Indian FAV serotype 4 isolates AD: 411 persisted in the caecal tonsils and livers of the birds up to 15 dpi, which was detected by FAT and virus isolation. The age of chicken, breed/line and immune competence are some of the factors to be considered to influence the persistence of virus in IHP affected chickens. Strong depletion of lymphocytes in the bursa of Fabricuous, thymus, spleen, and gross atrophy of these organs observed by us is also reported in an experimental study with FAV serotype 8 isolated from IBH of chickens 22 Pst I and Bam HI genetic profiles of the viruses in this study suggested that 1989 outbreak of IHP/ hydropericardium syndrome in Angara Goth of Pakistan was the probable origin of early 1994 outbreak of IHP in Jammu and Kashmir, India. As all the six FA V serotype 4 isolates in this study exhibited close antigenic relationship, and displayed identical RE profiles and AD: 411 isolate was able to produce IHP experimentally and also showed close antigenic relationship with other five viruses. Thus, it is suggested that it may be suitable candidate for production of killed CEH culture based vaccine to adopt preventive measures in India. Different serotypes of group I FAV have been reported to induce IHP in chickens either naturally or experimentally in different parts of the world for example, serotypes 2, 4, 8 in JapanlS, FA V serotype 8 in Mexic0 23, FAV serotype 12 in USA I5, FA V serotype 4 in Pakistan, Russia and South American countries 23 and FAV serotype 2 in GermanylD. Hence continuous surveillance for identification of possible involvement of a new FA V serotype in IHP is necessary to implement successful prevention and control programme for the disease across the country. Acknowledgement Financial support by CSIR, New Delhi is duly acknowledged. 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