Newcastle Disease Virus Vaccine Strains : Immunogenicity is not Influenced by ICPI

Intracerebral pathogenicity index (ICPI) and mean death time (MDT) were determined using commercial live vaccines against Newcastle disease available in Brazil. The ICPI profiles obtained for B1 vaccine strains were nonvirulent and varied from 0 to 0.19, and their MDT was 104-116 hours. The LaSota strains had an ICPI varying between 0.02 and 0.37 and MDT from 92 to 116 hours. ICPI and MDT for the Clone 30 were 0.11 and 104 hours, respectively. For Ulster vaccines, ICPI and MDT were 0 and >150 hours; for VG-GA was 0.03 and 140 hours; and for C2, 0.04 and >144 hours. Eye drop vaccination and IM challenge, at the 1 week and the 4 week, respectively, resulted in highest protection for B1 (95-100%) and LaSota (90-100%) strains. The variability in vaccine ICPI did not interfere with immune response and all vaccines provided similar protection. All vaccines were considered non virulent and were classified as lentogenic according to the immunobiological product standards.


INTRODUCTION
The Newcastle disease virus (NDV) or avian paramyxovirus serotype 1 (APMV-1) is a RNA virus belonging to the genus Avulavirus of the family Paramyxoviridae (Mayo MA. 2002a;Mayo MA 2002b).It is an important pathogen that can affect commercial poultry producers worldwide by producing outbreaks, and resulting in trade barriers (Alexander et al., 1997).The disease is economically important, since it causes high morbidity and mortality, reduces egg production, deteriorates egg quality, and impairs live performance.
The importance and impact of a NDV isolate is directly related to its virulence.Laboratory tests were developed by Hanson & Brandly (1955) proposed the classification of NDV isolates upon allantoic inoculation using mean dead time (MDT) as "velogenic", "mesogenic", and "lentogenic", based on chicken embryo mortality at <60 hours, 60-90 hours, or >90 hours, respectively.Other tests designed to differentiate strains directly assess clinical signs or death in infected birds, calculating a pathogenicity index.The most widely used test is the intracerebral pathogenicity index (ICPI) (Hanson, 1980).NDV infection is currently defined as a notifiable disease if the virus has an ICPI of 0.7 in day-old chicks (Gallus gallus domesticus) (OIE, 2000b).An APMV-1 virus that does not meet the OIE definition for causing ND is referred to as a lowvirulence APMV-1 or NDV.
Efforts for ND prophylaxis in broiler chickens in Brazil are focused on the active immunization by the use of live lentogenic vaccines.The virus strains most commonly used in vaccines are the La Sota (Goldhaft, 1980) and the B1 strains (Hitchner & Johnson, 1948), as well as viruses from the asymptomatic enteric pathotype, which are usually based on the We are thankful to the Brazilian Ministry of Agriculture for authorizing publication of this paper.We would like to thank the coordinating bodies of Lanagro/SP, the Animal Laboratory, as well as the coordination of Veterinary Products.We also thank the Poultry Disease Sector staff, particularly Maria do Livramento de Jesus Nascimento and Jaíra Alves dos Santos, for their expert technical help, and Luiz Carlos da Silva Porto, for assisting in animal care.C.W.A. and F.R.S. acknowledge the funding provided by CNPq (National Council of Scientific and Technological Development, Brazil).We thank Etna Lima Macário, translator at the Research Commission (FCM-Unicamp), for collaborating with the English review.
V4, VG-GA or Ulster 2C viruses.These viruses are selected by manufacturers in order to improve vaccine immunogenicity or to enable their use by a particular method of application (Alexander et al., 2004).Some lentogenic vaccines have been cloned (Clone 30), selecting a virus which produces less vaccine reactions than a La Sota-like virus, with superior immunogenicity as compared to HB1-like viruses (Alexander et al., 2004).
In Brazil, as well as in the European Union, legislation defines the pathogenicity of viruses allowed to be used in vaccines.The virus seed from live vaccines must be tested, and their ICPI must have values lower than 0.4, while the seed of the inactivated virus used in vaccines must have an ICPI value lower than 0.7 (CEC, 1993).In Brazil, only lentogenic strains with ICPI <0.4 are allowed in commercial vaccines.A similar legislation was adopted by the Organization for Animal Health (OIE, 2004) and also in Brazil, as described in Regulation # 07/06 (BRAZIL, 2006).Brazilian law requires that vaccines used to immunize poultry against ND are produced from strains with low pathogenicity.The quality antigen of the viruses used in vaccines is of paramount importance to provide adequate immune response against NDV.
This study was carried out to determine if the profile of the Brazilian vaccines is in accordance with that described by the Organization for Animal Health -OIE (2004), and if there is a correlation between different ICPI/MDT and protection conferred by the same strains.

MATERIALS AND METHODS
Facilities.The experiment was carried out at the Poultry Health Sector of Lanagro/SP.The efficiency test, using pathogenic virus challenge, was conducted at biosafety level 3.
Virus vaccines.The commercial lyophilized vaccines, manufactured in Brazil or imported, were prepared with the strains (B1, La Sota, Ulster 2C, Clone 30, VG-GA, and C2).The titer was determined using the technique described for thermostability titration (Simi et al., 1970;Orsi et al., 2009, in press), and varied between 105.50 and 106.70.The vaccine vials were reconstituted in proper dilution, as recommended by the manufacturers, and strains were propagated in embryonated SPF chicken eggs.
Vaccine virus propagation in embryonated SPF chicken eggs.The vaccine and reference strains were replicated by inoculating embryonated SPF eggs (9-11 days old), via allantoic cavity.Embryos that died within 24 hours were discarded, and the remaining were tested for hemagglutination (HA) activity in the allantoic fluid, according to the technique described in Regulation # 07/06 (Brazil, 2006).The fluids containing HA activity were pooled, and stored at -80ºC until use.
Birds.One hundred and seventy-five unsexed dayold chickens derived from specific pathogen-free (SPF) eggs (Granja Rezende, Brazil), from flocks known to be free from antibodies against NDV were used in the experiments.Birds were housed in nine isolation units (20 per unit), operated under negative pressure with filtered air intake and exhausted air.Isolation units were placed in a high containment facility (biosafety level 3) of Lanagro/SP.In addition, ten one-day-old chickens, housed under similar conditions, were maintained as controls.Five one-day-old chickens were used as serum source to confirm the negative NDV serological status of the chickens at the beginning of the experiment.
Experimental design.Tests were carried out with the most common NDV strains used by the poultry industry (La Sota and B1), produced by four different manufacturers.One hundred sixty chickens were distributed into eight groups according to vaccine manufacturer and vaccine strain, housed in isolation units, and vaccinated once at seven days of age via eye drop.Each group (n=20) was vaccinated with one given commercial vaccine.Four vaccines were manufactured with La Sota strain and four with B1 strains by 8 different manufacturers.
One unit with ten birds was kept as unvaccinated control.The lyophilized vaccine was reconstituted using PBS at pH 7.2 in the proportion of 30 mL/1000 dose vaccine, and administered via eye drop dose, according to the methodology of Paulillo (1984Paulillo ( , 1989)).Twentyone days after vaccination, all groups (vaccinated and unvaccinated) were challenged by intramuscular injection (IM).After the challenge, birds were observed for clinical signs and daily mortality for 10 days.Resistance to challenge was expressed as percentage of total protection, and refers to the absence of clinical signs (morbidity) and death in 90% of the vaccinated and challenged birds, and to the presence of clinical signs and/or mortality in less than 90% of the challenged group.
Biological characterization: Intracerebral pathogenicity index (ICPI).ICPI was obtained using the in vivo test described in Regulation # 182/94 (Brazil, 1994).The World Organization for Animal Health (OIE, 2004) defines pathogenic Newcastle disease virus strains as those presenting an IPIC ≥0.70, and Newcastle disease is an infection of birds caused by the avian paramyxovirus serotype 1 (APMV-1) that meets the following virulence criterion: it has an ICPI ≥ 0.70 in day-old chicks (Gallus gallus).

Pathogenicity test of the ND vaccine strains.
Seven B1 strain vaccines, six La Sota strain vaccines, and the strains Ulster, VG-GA, Clone 30, and C2 were tested.
The biological characterization of the live vaccine strains showed that the ICPI of B1 strains ranged from 0.0 to 0.19 and its MDT, between 104 and 116 h (Table 1).The ICPI of the La Sota strain ranged from 0.02 to 0.37, and MDT varied between 92 and116 h (Table 2).The ICPI and the MDT were 0.11 and 104h for the Clone 30 strain, respectively; 0 and > 150h for the Ulster strain,0.03and 140h for the VG-GA strain, and 0.04 and >144h for the C2 strain (Table 3).The biological characterization of the ND vaccine strains includes ICPI and MDT determinations.All vaccine viruses were classified as lentogenic strains, according to their MDT values, and the embryos remained alive for more than 90 hours.ICPI values ranged from 0.0 to 0.37, characterizing the vaccine virus are lowly virulent or as nonpathogenic strains.Protective efficacy of the different ND vaccine strains.The B1 vaccine strain groups had higher numbers of survivors and, independently from manufacturer, the protective efficacy varied between 95 and 100 %.Regarding the La Sota vaccine strains, the protective efficacy varied from 90 to 100 % (Table 4)., 1993).These guidelines were adopted by OIE (2000b).The ICPI results for all vaccines used in Brazil, as observed in the present study, complied with the OIE requirements (ICPI ≤ 0.37) relative to nonpathogenic strains.
The main goal of live vaccines is to establish an infection status in a flock, preferably in each bird at the time of application.Individual bird treatments, such as intranasal instillation, eye drop, and beak dipping, are often used with lentogenic vaccines.
The vaccine administration method via eye drop is probably the most effective for live lentogenic vaccines.It ensures that the vaccine reaches individual birds and, consequently, the obtained titers are usually uniform throughout the flock.
The results obtained with the pathogenic NDV strain in 28-day-old SPF birds in the present experiment showed satisfactory efficacy, with results equal to or above the minimum required by the Brazilian legislation (Brazil, 2006).
This study confirms that all live vaccines strains used in Brazil are lentogenic, based on mean death time (MDT) having chicken embryo mortality at > 90 hours (Hanson and Brandly, 1955.In conclusion, it was confirmed that the vaccine strain B1 (ICPI between 0 and 0.13) and the vaccine strain La Sota (ICPI between 0.02 and 0.37) are efficient up to their validity period of 24 months, producing results equal to or above the minimum required by the Brazilian legislation.
The results obtained in the present study showed that the intracerebral pathogenicity index differences among Newcastle disease virus strains do not interfere with the immune response in Gallus gallus domesticus.

CONCLUSION
All vaccines used in Brazil were considered nonvirulent, and were classified as lentogenic according to the standards established for immunobiological products.The differences in intracerebral pathogenicity index among Newcastle disease virus strains do not interfere with the immune response in Gallus gallus domesticus.
to 100%, all vaccines protected against the challenge.Unvaccinated SPF chickens developed clinical ND clinical signs and/or death, and 0% protection against ND appeared by the 3 rd day after challenge Relationship between efficacy test and ICPI.The relationship between the efficacy test and the intracerebral pathogenic test (ICPI) of two vaccine strains (La Sota and B1) showed that B1 vaccine strains had an ICPI varying from 0 to 0.13 and its protective efficacy varied between 95 and 100%.La Sota vaccine strains presented an ICPI between 0.02 and 0.37 and protective efficacy from 90 to 100% (Table 5).

DISCUSSION
There is little information on the classical biological characteristics (ICPI and MTD) of vaccines used in Brazil.According to Nunes et al. (2002), who performed a comparative morphometric analysis of vaccine virulence of NDV lentogenic strains (La Sota, Ulster, and VG-GA), La Sota and Ulster presented the same virulence on the third day after vaccination, and both caused higher swelling of tracheal mucosa than the VG-GA strain.The results obtained with chickens vaccinated with La Sota strains in the present study are consistent with a previous experiment quantifying tracheal swelling (Jorge et al., 1998).Newcastle disease is defined by the OIE (OIE, 2000a(OIE, , 2004) ) as an avian infection caused by the avian paramyxovirus serotype 1 (APMV-1), which meets the following criterion for virulence: intracerebral pathogenicity index (ICPI) of 0.7 or greater in day-old SPF chicks (Galllus gallus domesticus).
The use of vaccines in the European Community is allowed when the seed vaccine to be tested shows an ICPI <0.4 (10 7 EID 50 / bird), or <0.5 (10 8 EID 50 /bird).
The OIE Standard Committee recommends that a vaccine must have an ICPI <0.7 in order to meet the estimated interlaboratory variability.A safety margin is allowed, and the seed strains used for vaccination

Table 1 -
Pathogenicity index values obtained for commercial NDV B1 vaccine strains.

Table 2 -
Pathogenicity index values obtained for commercial NDV La Sota vaccine strains.

Table 3 -
Pathogenicity index values obtained for commercial NDV vaccine strains.

Table 4 -
Challenge results for NDV "São João do Meriti"* strain of SPF birds vaccinated at 7 days of a 28th days olddays, use of eye drop by vaccination methods.
***Percentage of surviving birds that did not display clinical signs of disease.Calculated as: 1-(n. of affected chickens + n. of dead chickens)/n. of immunized chickens X100.Considering that their protection ranged from 90

Table 5 -
Protective efficacy and ICPI of different vaccine strains.