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The following technical article is related to the event:
XXII Latin American Poultry Congress 2011

Recombinant vaccine for poultry against Newcastle Disease

Development of a recombinant vaccine and its efficacy in a trial with broilers

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Summary

With the objective of achieving a more effective control of problems caused by Newcastle disease virus (NDV) in specific regions, a live recombinant vaccine expressing the gene of the highly pathogenic protein hemagglutinin-neuraminidase (HN) from a virus of genotype V (strain P05) was developed. This was done with the use of reverse genetics. The velogenic virus used in the development of this vaccine was genetically modified at the cleavage site of the fusion protein in order to change the highly pathogenic genome to one similar to LaSota strain and thus decrease its virulence. A field experiment was conducted to evaluate vaccine protection in a broiler flock and its effects on production parameters compared to the application of a LaSota strain vaccine. The results for the group vaccinated with the experimental virus showed lower mortality, younger market age, better feed conversion rate and improved productivity index. More over, viral shedding was determined, which was lower in the group receiving the experimental vaccine.
Key Words: Newcastle disease, Homologous, Genotype V, Recombinant virus, Fusion protein.

Introduction

The Newcastle disease virus in its velogenic form continues to be a serious problem in the poultry industry worldwide. Biosecurity and vaccination measures are the more effective control methods currently available. However, the antigenic and genetic diversity of the virus has put a phylogenetic distance of the current vaccine strains from the recent  velogenic isolates found in Central and South America (Miller et al., 2010), in some U.S. regions (Peedersen et al., 2004 ) and in Mexico pertaining to the genotype V, Class II (Perozo et al., 2008). Such heterology could facilitate the evolution of virulent ND virus (Miller et al., 2007). In one study, Lucio et al. (2007) demonstrated the phylogenetic distance between field strains isolated in Mexico and the La Sota strain, widely used in this and other countries. Various studies have been conducted to compare the effectiveness of frequently used vaccines and vaccines with strains homologous to the challenges (Miller et al., 2009). There is recent evidence that the use of vaccines homologous to the challenge virus may reduce viral shedding or excretion. Miller et al. (2007) showed that a vaccine homologous to the challenge strain (CA02 of genotype V) significantly reduced oral viral excretion more than inactivated heterologous vaccines with strains such as B1 and Ulster (genotype II and I, respectively). Hu et al. (2009) showed similar effects against viruses of genotype VII.

The objective of this study was to develop a vaccine with a recombinant virus (chimera) to express the gene for hemagglutinin-neuraminidase of a genotype V (class II) virus by a change in the genetic sequence at the cleavage point of the fusion protein and determine its efficacy against mortality to a field challenge, and its effects on production parameters in a broiler flock, as well as viral excretion or shedding.

Materials and Methods

Development of recombinant vaccine strain recP05
The genome in the form of La Sota strain RNA was provided by the Biology Lab of Investigación Aplicada S. A. de C. V. P05 strain was isolated in 2005 by Investigación Aplicada S. A. de C. V. and its genome in the form of RNA was provided by the Biology Lab of the same company.

We used the method of reverse transcriptase and specific primers were designed for amplification and sequencing of the virus. Sequential change was made at the cleavage site of the F protein of P05 strain to the same sequence of La Sota virus. Lastly, three plasmid vectors for pIASAP, pIASANP and pIASAL expression for genes P, NP and L of P05 virus for proteins that form the RNA polymerase complex were built and the virus in Hep 2 cells after being infected with "Vaccinia" virus of Ankara modified to express the RNA polymerase of bacteriophage T7 was recovered. The final construction of the recombinant virus genome is presented in Figure 1.

Figure 1. Construction of the virus recP05 strain.

Use of the vaccine with recP05 strain in a broiler flock
The protection provided by the recombinant vaccine of genotype V recP05 in a broiler farm was evaluated, where a total of 299,943 Ross breed chickens were used, divided into 2 groups: Group A of 96,869 chickens and Group B of 203,074 chickens. Birds of both groups received the same medical and animal husbandry management. Therefore, all birds were vaccinated with a bivalent vaccine of inactive ND virus (genotype V) and Avian Influenza (A/Chicken/Mexico/232/94CPA) by emulsion on day 1 and 10 of age. At 10, 20, and 31 days of age, Group A birds received the experimental vaccine of live ND virus, genotype V strain (recP05), while Group B birds received a live ND virus vaccine, genotype II strain (La Sota). The same management, biosecurity and feeding practices for both groups were performed at the farm, and the following flock production parameters log was carried out: total mortality rate, mortality percentage, average market age, average market weight, feed conversion, daily weight gain (DWG) and productivity index. Tracheal swabs were collected at 4, 5, and 6 weeks to detect viral particles by real-time PCR reaction (Polymerase Chain Reaction). Virus genetic isolation and sequencing was performed.

Results & Discussion

Percent mortality (%)
Group A birds vaccinated with live virus of genotype V strain showed lower mortality (4.76%), with a difference of 12.17% compared to Group B (16.93%) vaccinated with a strain of genotype II (Table 1).

Average market age (days)
Group A birds with live virus vaccine of genotype V strain resulted in 0.8 less days to market (42.1 days) compared with Group B birds vaccinated with live virus of genotype II strain (42.9 days ) (Table 1).

Average market weight (grams)
Group A vaccinated with experimental recP05 live virus vaccine had 98 g less of market weight (1,965 g) compared to Group B vaccinated with live virus of genotype II strain (2,063 g). (Table 1).

Feed conversion
Group A vaccinated with live virus vaccine of Genotype V strain showed a 0.056 points better feed conversion, or 56 fewer grams of feed consumed per kilogram produced (1.902) compared with Group B vaccinated with live virus of genotype II strain (1.958).

Daily weight gain (grams)
Group A vaccinated with live virus of Genotype V strain had 1.34 g less ADWG at the end of the growing period (46.44 g) compared to Group B vaccinated with live virus of genotype II strain (47.78 g). (Table 1)

Productivity index
Group A vaccinated with live virus vaccine of Genotype V strain showed a better index (214.40), compared with Group B vaccinated with live virus of genotype II strain (202.68). (Table 1)

Table 1. Flock production parameters and difference in results between Group A and Group B.

Production variable

Group A

Group B

Difference

Group A vs. Group B

No. of birds

96,869

203,074

 

Average market age (days)

42.1

42.9

0.80 days

Average market weight (g)

1,965

2,063

- 98 g

Feed conversion

1.902

1.958

- 0.056

Daily weight gain (g)

46.44

47.78

- 1.34 g

Final mortality %

12.17 a

16.93 a

- 4.76%

Productivity index

214.40

202.68

11.72

At week 4, the real-time PCR test was positive in both groups. However, a greater viral particles titer was observed in Group B vaccinated with La Sota strain, with a log difference (Table 2). Genetic sequencing was performed and the genotype V virus was identified. Viral isolation revealed the presence of a velogenic virus.

Table 2. Viral shedding. Results of real-time PCR of viral particles detection in pharyngeal exudate.

 

Group A

Group B

Result

Titer DI/mL

Result

Titer DI/mL

Week 4

Positive

6.3X105

Positive

2.04X106

Week 5

Negative

Not detectable

Negative

Not detectable

Week 6

Negative

Not detectable

Negative

Not detectable

The results obtained agree with the observations of Miller et al. (2007) who showed that vaccines made with virus homologous to challenge virus decreased viral excretion more than the heterologous vaccines, so they suggest that vaccines formulated to be phylogenetically closer to the potential challenge virus may facilitate control of ND by reducing transmission from infected birds. Vaccines used today generally protect against morbidity and mortality in field challenges. However, according to van Boven et al. (2008), in addition to this. the effectiveness of vaccines must be determined, because of its ability to reduce viral excretion.

Conclusions

In recent years, there has been increased interest in updating ND vaccine strains in order to provide greater protection to flocks by controlling the recirculation of virus in farms by viral shedding, that is usually detected in birds receiving vaccines with heterologous strains. The use of vaccines made with a recombinant virus of genotype homologous to a field virus showed that it is possible to provide an adequate protection against mortality and provide better control of viral excretion, as well as improve production parameters of a broiler flock. This shows that benefits obtained with genetic technology advances and the analysis of the situation in the field are consistent, in relation to a disease that continues to concern both the industry and the scientific community seeking to improve control methods.

References

Hu S, Ma H, Wu Y, Liu W, Wang X, Liu Y, Liu X. 2009. A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics. Vaccine 27:904-910.

Lucio E, Morales A, Ortega R, Rodríguez A, Absalón A. 2007. Características de los virus de la enfermedad de Newcastle que circulan en México y sus repercusiones en protección y persistencia en las zonas afectadas. Tehuacán, Pue., México. Investigación Aplicada SA de CV e Instituto de Biotecnología del IPN, Tlaxcala, México.

Miller PJ, King DJ, Afonso CL, Suarez DL. 2007. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 25:7238-7246.

Miller PJ, Estevez C, Yu Q, Suarez D, King D. 2009. Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses. Avian Dis. 53:39-49.

Miller PJ, Lucio E, Afonso CL. 2010. Newcastle disease: Evolution of genotypes and the related diagnostic challenges. Infection Gen. Evol.10:26-35.

Pedersen JC, Senne DA, Woolcock PR, Kinde H, King DJ, Wise MG, Panigrahy B, Seal BS. 2004. Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003 outbreak in California and other recent outbreaks in North America. J. Clin. Microbiol. 42:2329-2334.

Perozo F, Merino R, Afonso CL, Villegas P, Calderon N. 2008. Biological and phylogenetic characterization of virulent Newcastle disease virus circulating in Mexico. Avian Dis. 52:472-479.

Van Boven M, Bouma A, Fabri TH, Katsma E, Hartog L, Koch G. 2008. Herd immunity to Newcastle disease virus in poultry by vaccination. Avian Pathol. 37:1-5.

 

 

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