Introduction
Avian adenovirus caused the first clinical disease in the face of an outbreak of respiratory disease in quails in 1950 (Oslon, 1950) and it was identified as a quail bronchitis virus. Its name is derived from ‘adeno’ -a Greek word ‘aden’ or ‘adenos’ meaning gland, because the first human adenovirus isolated from human adenoid tissue. Avian adenoviruses are known to cause various disease conditions in birds, marine life, animals and humans. Infections caused by the adenoviruses may be asymptomatic or can cause severe pathological conditions and sometimes high mortality.
In 1987, a new and different disease condition was observed in broiler chickens at Angara Goth near Karachi in Pakistan, which was named Angara disease. The disease was destructive and caused the death of 100 million broiler bird in Pakistan in less than two years (Jaffery, Reference JAFFERY1990; Cowen et al., Reference COWEN, LU, WEINSTOCK and CASTRO1996). The disease is known by several names, including the Angara disease in Pakistan, Lichee disease in India (because of the heart floating in fluid similar to de-shelled lichee fruit; Gowda and Satyanarayana, Reference GOWDA and SATYANARAYANA1994), inclusion body hepatitis hydropericardium syndrome (IBH-HPS) (Abdul-Aziz and Al-Attar, Reference ABDUL-AZIZ and AL-ATTAR1991), hydropericardium hepatopathy syndrome (Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997), or hydropericardium hepatitis syndrome (HHS) (Ganesh et al., Reference GANESH, SURYANARAYANA and RAGHAVAN2002). Disease outbreaks have been reported not only in India and Pakistan but also in the Middle East, Japan, Ecuador, Peru, Chile and Mexico (Abe et al., Reference ABE, NAKAMURA, TORO, MASE, SHIBAHARA and YUASA1998; Hess et al., Reference HESS, RAUE and HAFEZ1999; Toro et al., Reference TORO, GONZALEZ, CERDA, HESS, REYES and GEISSEA2000).
Hydropericardium syndrome (HPS)
Khawaja et al. (Reference KHAWAJA, AHMAD, RAUF, ZULIFQAR, MAHMOOD and HASAN1988) and Jaffery (Reference JAFFERY1988) reported a pathological condition in broiler chickens aged three to six weeks old with peculiar lesions in the heart and liver (Balamurugan and Kataria, Reference BALAMURUGAN and KATARIA2004). The first epidemic of HPS was reported in 1987 from Angara Goth, Karachi (Jaffery, Reference JAFFERY1988; Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989), although sporadic cases were recorded as early as 1985 (Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989). In India HPS was reported in some parts of Jammu and Kashmir, Punjab and Delhi in 1994 (Gowda and Satyanarayana, Reference GOWDA and SATYANARAYANA1994). Initially it was hypothesised that HPS was caused by toxicity or by a nutritional disorder, however later an infectious agent was suggested as the probable cause (Abdul-Aziz and Al-Attar, Reference ABDUL-AZIZ and AL-ATTAR1991). Based on the successful isolation of an agent from the liver, heart, and kidneys, and transmission of the disease after subcutaneous inoculation with infected liver homogenate, the aetiological agent was tentatively designated as an adenovirus (Khawaja et al., Reference KHAWAJA, AHMAD, RAUF, ZULIFQAR, MAHMOOD and HASAN1988). Subsequent studies demonstrated typical icosahedral particles of adenovirus in the purified liver homogenate extracts by negative-staining electron microscopy. Additionally, the presence of intranuclear basophilic inclusion bodies in the liver cells supported the view that the disease is caused by adenovirus (Mazaheri et al., Reference MAZAHERI, PRUSAS, VOSS and HESS1998).
The most important economic losses associated with the disease have been reported in three to five weeks old broilers; however, breeders and layers may be affected (Christensen and Saifuddin, Reference CHRISTENSEN and SAIFUDDIN1989; Akhtar, Reference AKTHAR1994). Adenovirus isolates associated with HPS, obtained recently from Chile, have been identified both by virus neutralisation tests and by restriction endonuclease analysis of particular DNA fragments, as members of the fowl adenovirus serotype 4 (FAV 4) (Cowen et al., Reference COWEN, LU, WEINSTOCK and CASTRO1996). Other adenoviruses obtained from recent HPS outbreaks in South America have also been identified as FAV 4 (Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989). However, although HPS has been associated with FAV strains, there is conflicting evidence concerning their role as primary etiologic agents in the induction of HPS under field conditions. Some authors have underlined their role as primary etiologic agents (Aghakan and Pattison, Reference AGHAKAN and PATTISON1974; Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989), whereas others have supported the hypothesis that most FAV strains act as secondary pathogens in association with immunosuppressive agents (Akhtar et al., Reference AKHTAR, ZAHID and KHAN1992; Abdul-Aziz and Hasan, Reference ABDUL-AZIZ and HASAN1995; Benkő et al., Reference BENKŐ, HARRACH, RUSSELL, ADAIR, DE JONG, HESS, JOHNSON, KAJON, KIDD, LEHMKUHL, LI, MAUTNER, PRING-AKERBLOM and WADELL2005).
Aetiology/disease
Initially it was suspected that HPS was caused by a nutritional deficiency or ingestion of toxins. The possible causes and factors investigated included fats, sodium chloride, mycotoxins, chlordane, polychlorinated biphenyls and phytotoxins, all of which are associated with hydro-pericardium (Jaffery, Reference JAFFERY1988; Qureshi, Reference QURESHI1989). All of the attempts to reproduce the disease via feed failed, even though these attempts included samples of feed from infected farms, feed containing mycotoxins or both (Anjum, Reference ANJUM1988; Reference ANJUM1990). Anjum et al. (Reference ANJUM, SABRI and IQBAL1989) and Ahmad et al. (Reference AHMAD, AFZAL, MALIK, HUSSAIN and HANIF1989) finally reproduced the disease by injecting bacteria from free liver homogenate in an experiment with young broiler chickens and from this proved that an infectious agent was the possible cause.
Further studies revealed that the adenovirus was associated with the disease condition, this being incriminated by the demonstration of basophilic inclusion bodies in the hepatocytes of affected birds (Niazi et al., Reference NIAZI, KHAN and SIDDIQUE1989; Afzal et al., Reference AFZAL, MUNEER and STEIN1991; Gowda, Reference GOWDA1994). Electron microscopy demonstrated discrete icosahedral virions in the purified liver extracts by negative staining (Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989). All the field isolates collected from outbreaks of HPS have been serotyped as FAV-4 using standard anti sera to 12 different serotypes (Jadhao et al., Reference JADHAO, KATARIA, VERMA and SHAH1997) and identified as HPS viruses (Vairamuthu et al., Reference VAIRAMUTHU, RAJA, BALASUBRAMANIAN and MANOHAR2002).
For certain South American countries and in Pakistan, in almost all the cases associated with hydropericardium, FAV-4 is solely responsible as the causative agent for the specific disease condition (Voss et al., Reference VOSS, VIELITZ, HESS, PRUSAS and MAZAHERI1996). In Pakistan, an adenovirus (K31/89), isolate of field outbreak of HPS (Voss, Reference VOSS1989), was identified as fowl adenovirus serotype 4 (FAV-4). European serotype 5 (FAV-8), which is associated in the production of hydropericardium with inclusion body hepatitis (IBH) in Mexico, has also been reported (Shane and Jaffery, Reference SHANE and JAFFERY1997). These two conditions have appeared at the same time in many outbreaks hence the disease is called inclusion body hepatitis hydro-pericardium syndrome (IBH-HPS).
The IBH-HPS was reproduced in specific pathogen free (SPF) chickens by the use of isolated and purified viruses from field outbreaks (Cowen et al., Reference COWEN, LU, WEINSTOCK and CASTRO1996), thus proving the association with FAV-4. The avian adenoviruses are non-enveloped DNA viruses with a diameter of 70 to 90 nm. Genomic DNA comprises 11 to 13% of the virion and accessory proteins stabilise the viral capsid, and the virus has a characteristic icosahedral symmetry (McFerran, Reference McFERRAN1998). The virion capsid is composed of a single layer containing 252 capsomers. It has 240 capsomers or hexons which are 8 to 10 nm in diameter and make up the major soluble component harvested from cell cultures (McFerran and McConnel, Reference McFERRAN and McCONNEL2003). The genome is composed of a single, linear, non-segmented double-stranded DNA molecule of 43 kbp. Replication of the virus starts after 10 hours post infection in the nucleus of the host cell, producing basophilic intra-nuclear inclusion bodies (Villamandos et al., Reference VILLAMANDOS, HERVAS, CARRASCO, CABALLERO and SIERRA1996). Several disinfectants are resisted by Adenoviruses and relatively tolerant to pH changes and heat. Long time exposure with iodophor and aldehyde disinfectants seems to be effective in controlling the adenovirus. Complete inactivation of the virus requires 20 days of infected chicken carcasses composting (Senne et al., Reference SENNE, ANIGRAHY and MORGAN1994).
Microscopic and gross lesion
Avian adenoviruses were placed in a very varied group of pathogenic microorganisms which cause a diversity of problems for poultry. Among various emerging diseases of poultry, the avian adenoviruses have been incriminated as aetiological agents for different clinical conditions in poultry (Fadly and Winterfield, Reference FADLY and WINTERFIELD1973; Rosenberger et al., Reference ROSENBERGER, ECKROADE, KLOPPS and KRAUSS1974). A range in incubation periods, from 5 to 17 days, with an average of 10 days, has been reported in experimental trials (Akhtar et al., Reference AKHTAR, ZAHID and KHAN1992). In a study by Ganesh et al. (Reference GANESH, SURYANARAYANA, RAGHAVAN and GOWDA2001) it was observed that the incubation period ranges from two to five days after inoculation of homogenate and purified virus.
Vertical and horizontal transmission plays a vital role in causing IBH-HPS. Vertical transmission has been observed in progeny from breeder flocks infected with avian adenovirus serotypes 8 and 4. Horizontal transmission has been confirmed in young broiler chicks, which, when in contact with infected chicks, can die of acute IBH-HPS.
In natural outbreaks of IBH-HPS, the infected birds may not show any clinical signs (Jaffery, Reference JAFFERY1988), excluding sudden onset of high mortality rate (up to 80%) in broiler birds aged three to six weeks old, and low mortality in layers (about 10%), associated with hydropericardium (Chandra et al., Reference CHANDRA, SHUKLA and and KUMAR2000). The consistent and predominant gross lesions at necropsy are seen as hydropericardium in more than 90% of the affected birds (Anjum et al., Reference ANJUM, SABRI and IQBAL1989; Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989; Qureshi, Reference QURESHI1989; Gowda and Satyanarayana, Reference GOWDA and SATYANARAYANA1994). This condition is characterised by the presence of a watery or jelly like fluid in the pericardial sac, which may be amber green in colour or colourless. The accumulated fluid is usually in the range of 3 to 20 ml in quantity and has a pH of 7 (Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989; Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997). The heart appears misshapen and flabby (Anjum et al., Reference ANJUM, SABRI and IQBAL1989), with its apex floating in the pericardium (Kumar et al., Reference KUMAR, CHANDRA, SHKUKLA, AGRAWAL and KUMAR1997). The tissue fat may exhibit yellowish discoloration and petechial haemorrhages (Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997). The liver is mottled and has focal necrotic patches, discoloured pale, the kidneys appear yellowish and friable, having deposits of urates in the tubules and ureters and oedematous and congested lungs are seen (Nakamura et al., Reference NAKAMURA, MASE, YAMAGUCHI, SHIOBAHARA and YUASA1999).
The severity of the lesions and mortality appears to be higher in immunosuppressed birds (Nakamura et al., Reference NAKAMURA, SHOYAMA, MASE, IMADA and YAMADA2003). The liver was recognised as the most important organ in IBH-HPS which showed changes upon histological study, such as mononuclear cell infiltration, multifocal small areas of coagulative necrosis with basophilic intranuclear inclusion bodies (INIBs) in liver cells bounded by clear halo, or as a filling in the whole enlarged nucleus (Anjum et al., Reference ANJUM, SABRI and IQBAL1989; Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989; Gowda and Satyanarayana, Reference GOWDA and SATYANARAYANA1994; Abdul-Aziz and Hasan, Reference ABDUL-AZIZ and HASAN1995; Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997; Kumar et al., Reference KUMAR, CHANDRA, SHKUKLA, AGRAWAL and KUMAR1997; Nakamura et al., Reference NAKAMURA, MASE, YAMAGUCHI, SHIOBAHARA and YUASA1999). These changes were confirmed through transmission electron microscopic studies of the liver cells (Chandra et al., Reference CHANDRA, SHUKLA, KUMAR and GARG1997). The lungs showed oedema, congestion and infiltration by inflammatory cells (Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997; Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989) and there were haemorrhagic exudates in both alveoli and bronchi (Kumar et al., Reference KUMAR, CHANDRA, SHKUKLA, AGRAWAL and KUMAR1997) and a moderate diffuse infiltration of macrophages into the pulmonary parenchyma (Nakamura et al., Reference NAKAMURA, MASE, YAMAGUCHI, SHIOBAHARA and YUASA1999). Histopathological examination of the heart revealed mononuclear cell infiltration; sever vascular changes (Anjum et al., Reference ANJUM, SABRI and IQBAL1989) massive oedema haemorrhages (Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997) and degenerative changes (Gowda and Satyanarayana, Reference GOWDA and SATYANARAYANA1994). The kidneys showed marked swelling of the tubular epithelium, extensive haemorrhages and necrosis (Abdul-Aziz and Hasan, Reference ABDUL-AZIZ and HASAN1995; Asrani et al., Reference ASRANI, GUPTA, SHARMA, SINGH and KATOCH1997).
Vaccine
IBH-HPS is a highly infectious disease and the virus is very resistant to disinfectants. Vaccination against the disease is the only choice to protect birds from IBH-HPS. Immunisation of birds was previously carried out using autogenous formalin inactivated liver homogenate vaccines (Cheema et al., Reference CHEEMA, AHMAD and AFZAL1989; Chishti et al., Reference CHISHTI, AFZAL and CHEEMA1989; Ahmad et al., Reference AHMAD, MALIK, IQBAL, AHMAD and NAZ1990; Mashkoor et al., Reference MASHKOOR, HAMEED, AHMAD and QURESHI1994; Shane, Reference SHANE1996; Kumar et al., Reference KUMAR, CHANDRA, SHKUKLA, AGRAWAL and KUMAR1997). A formalin inactivated vaccine dose of 0.25 ml/bird at the age of 10-15 days gave variable results (Afzal and Ahmad, Reference AFZAL and AHMAD1990). However, a single dose of vaccination at the age of 15-18, was more helpful in preventing relapses at 35-40 days of age (Ahmad et al., Reference AHMAD, MALIK, IQBAL, AHMAD and NAZ1990).
An inactivated and freeze dried liver homogenate vaccine, re-suspended in normal saline can provide 100% protection, and was more effective than vaccines prepared from pellets obtained from ultracentrifugation and re-suspended in normal saline (Mashkoor et al., Reference MASHKOOR, HAMEED, AHMAD and QURESHI1994). Formalin inactivated vaccines have several drawbacks and the use of these has been discouraged. Ahmad and Hasan (Reference AHMAD and HASAN2004) reported that formalin inactivated, aqua based liver organ vaccines for hydropericardium syndrome are effective for control of the disease, while on the other hand contaminated vaccines are considered to play a major role in its geographical spread. In two experimental field trials, which involved 570,000 birds on 128 farms, the overall mortality ranged from 0.77 to 3.8% in vaccinated compared to 11.11 to 30% in unvaccinated birds (Ahmad et al., Reference AHMAD, MALIK, IQBAL, AHMAD and NAZ1990). In another study, the mortality in vaccinated birds against the disease was 0.52% compared to 5.34% in unvaccinated birds kept at similar premises. The vaccination of birds was deemed effective in the face of outbreak and significantly reduced mortality (Chandra et al., Reference CHANDRA, SHUKLA, KUMAR and GARG1997).
AAV 8/11 vaccine administered to broiler breeder progenies from 30 to 50-week old grandparents, gave adequately protection against challenge with AAV 8 and 11 serotypes and the Stanford strain, which was molecularly characterised as European serotype 9 (Alvarado et al., Reference ALVARADO, VILLEGAS, EL-ATTRACHE, JENSEN, ROSALES, PEROZO and PURVIS2007). Indian isolates from different field outbreaks of IBH-HPS in poultry farms from different geographical locations have been serotyped as FAV serotype 4 (Jadhao et al., Reference JADHAO, KATARIA, VERMA and SHAH1997) and the agent was characterised by restriction enzyme (RE) analysis of its genomic DNA (Jadhao, Reference JADHAO1998). Enzyme linked immunosorbant assay (ELISA) using an adenovirus free antigen did not fail to exhibit significant titres, nor did the cell mediate immune (CMI) response, as evidenced by delayed hypersensitivity reaction in the wattle, which showed a significant increase following vaccination (Noor-ul-Hassan et al., Reference AFZAL, HAMEED and KHAN1994). The PARC-1 isolate of HPS caused immunosupression that resulted in a reduced immunological response to Newcastle disease vaccine as compared to that in controls during an experimental study (Naeem et al., Reference NAEEM, NIAZI, MALIK and CHEEMA1995). Since the aetiology of the condition has almost been confirmed, immunodiagnosis by a variety of serological tests such as indirect haemagglutination (IHA), have been used (Hussain et al., Reference HUSSAIN, RASOOL, ZAHOOR and MANSOOR2003). Akhtar et al. (Reference AKHTAR, AHMAD, HAYAT, HUSSAIN and ASHFAQUE2000) used IHA to study the effect of coccidiosis on humoral immune response chickens vaccinated against hydropericardium syndrome (HPS). Agar gel precipitation tests (AGPT), counter immunoelectrophoresis (Kumar et al., Reference KUMAR, CHANDRA, SHKUKLA, AGRAWAL and KUMAR1997), the fluorescent antibody technique, various modification of ELISA (Balamurugan and Kataria, Reference BALAMURUGAN and KATARIA2004; Oberoi et al., Reference OBEROI, SINGH and SINGH1996) and the dot immunobinding assay (Naeem et al., Reference NAEEM, NIAZI, MALIK and CHEEMA1995) are all considered specific and reliable. Manzoor and Hussain (Reference MANZOOR and HUSSAIN2003) performed a reverse passive haemagglutination (RPHA) test for the detection of IBH-HPS virus using 1% suspension of sheep erythrocytes.
According to recent reports, formalin inactivated vaccines are unsuccessful in protecting broiler birds from this infectious disease. Liver organ vaccines showed significantly lower titres compared to chicken embryo adapted vaccines in broilers. The protection level of the vaccines has been estimated via clinical symptoms, mortality, gross and microscopic lesions and the histopathology of the liver (Mansoor et al., Reference MANSOOR, HUSSAIN, ARSHAD and MUHAMMAD2011). Oil emulsified vaccines induce greater antibody titres (Mashkoor et al., Reference MASHKOOR, HAMEED, AHMAD and QURESHI1994) as compared to formalin inactivated liver homogenate vaccines. An inactivated vaccine, prepared from liver homogenate extracted with chloroform and inactivated with formalin and adjuvanted with liquid paraffin, was highly effective against challenge in chickens aged four, five and seven weeks old (Zia et al., Reference ZIA, RAHMAN, SIDDIAQUE and SANDHU2001). Montanide adjuvanted vaccine was prepared and evaluated in broiler chickens against IBH-HPS by Aslam et al. (Reference ASLAM, HUSSAIN, MAHMOOD, KHAN and AKHTAR2012). The Montanide adjuvanted vaccine conferred 95% protection based on clinical signs, mortality and hydropericardium as compared to commercial oil based and formalin inactivated IBH-HPS vaccines.
Currently, formalin inactivated liver-based vaccines have been unsuccessful in protecting the Pakistan broiler industry from this disease (Mansoor et al., Reference MANSOOR, HUSSAIN, ARSHAD and MUHAMMAD2011). Owing to the infectious nature of IBH-HPS, the development of an appropriate vaccine in cell culture systems seem to be the better option, although it requires strict biosecurity and a high standard of hygiene and management.
More recently, vaccines propagated in cell culture systems and embryonated chicken eggs have been developed. Experimentally, a vaccine was developed by propagating the virus in chicken embryo liver cells and embryonated eggs by the allantoic route (Naeem et al., Reference NAEEM, NIAZI, MALIK and CHEEMA1995). Mansoor et al. (Reference MANSOOR, HUSSAIN, ARSHAD and MUHAMMAD2011) developed an embryo adapted and attenuated vaccine to protect broilers. A field isolate was successfully adapted to chickens embryos after four blind passages, and the adapted virus was passed serially 12 times more for attenuation. The prepared vaccine was trialled and groups of broilers were vaccinated with 16th passage attenuated virus vaccine and commercially available formalin inactivated vaccine. The antibody response, measured by ELISA was significantly higher (P<0.05) in the groups vaccinated with 16th passage attenuated viral vaccine as compared to the other group vaccinated with liver organ formalized vaccine at 7, 14 and 21 days post-vaccination. In a challenge protection assay, the group vaccinated with 16th passage attenuated vaccine had 94.73% protection, as evaluated on the basis of clinical signs, gross lesions on the liver and heart, histopathological lesions in the liver and mortality. The birds administered with the liver organ formalined vaccine demonstrated significantly lower (P<0.05) antibody titre by ELISA, with a protection level of only 55%. Unvaccinated control group birds showed high mortality (70%) and morbidity (84%). Naeem et al. (Reference NAEEM, NIAZI, MALIK and CHEEMA1995) found that the inactivated cell culture vaccine protected birds against the disease upon subcutaneous challenge with 1 ml of a 20% liver homogenate.
Many strains of IBH-HPS have been adapted onto primary cell cultures of chicken embryo origin and cytopathic effects have been observed. These primary cell cultures include chicken embryo fibroblast (CEF) cells, chicken embryo kidney (CEK) cells and chickens embryo liver (CEL) cells. Roy et al. (Reference ROY, KOTEESWARAN and MANICKAM2001) reported that the IBH-HPS virus was adapted onto Vero cells after four blind passages. It must be borne in mind that the primary cell culture system produces a low yield of virus and may also be contaminated with some extraneous viruses of avian origin (Muller and Becht, Reference MULLER and BECHT1982). The Vero cell line is an approved substrate for viral vaccine production. Lin et al. (Reference LIN, WU, SHIH and HO2002) characterised a Vero-cell line adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate. On passage on Vero cell line the enterovirus produced high viral titres and remained genetically stable. These features, such as high yield of virus, strong immunity and stability of passage, are enviable features in a prototype virus for the development of an efficient viral vaccine.
Newcastle disease virus (NDV) was adapted on a Vero cell line after five consecutive passages (Ahamed et al., Reference AHAMED, HOSSAIN, BILLAH, ISLAM, AHASAN and ISLAM2004). Infectious bursal disease (IBD) virus was similarly adapted and an attenuated vaccine was evaluated. Vero cell adapted and attenuated vaccines gave 100% protection based on the level of mortality (Rasool and Hussain, Reference RASOOL and HUSSAIN2006). The Vero cell culture adapted and attenuated vaccine conferred 100% protection in broiler chickens in a challenge protection study conducted at the Institute of Microbiology, University of Agriculture, Faisalabad; Pakistan (Ali, Reference ALI2013, unpublished data). IBH-HPS virus was adapted on a continuous cell line system. The adapted virus was attenuated and used as a vaccine candidate. The cell culture adapted and attenuated vaccine contained purified virus at a calculated dose with predictable immune response.
Conclusions
IBH-HPS is an important welfare and economic disease in commercial poultry farming. Identifying new methods of producing vaccines with a high protection rate is important in controlling the disease, and limiting its further geographical spread. Several new system for vaccine production have increased protection levels to much higher rates compared to older systems, and offer a suitable route for controlling the disease in commercial flocks.
