The presence of Infectious Bursal Disease virus (IBDV) from Latin American broiler and layer farms was examined. Suspect samples (FTA cards) from a number of Latin American countries were received and processed in the USA. During 2010 we received 187 samples, 86 of which were positive to reverse transcriptase-polymerase chain reaction (RT-PCR). The variable regions of viral protein 2 (VP2) from all positive samples were sequenced. Whenever identical viruses occurred in the same farm, such viruses were included only once in the phylogenetic analysis. The majority of the isolates (52%) were identified as variant IBDV strains. Classical and very virulent viruses represent 8.5% of the isolates. Vaccine-like viruses were identified in 31% of the samples. We will keep doing this survey in following years, which will serve as baseline for new epidemiological studies.
Key Words: Gumboro Disease, IBDV, Sequencing, Diagnostic, Latin America.
Infectious bursal disease is an acute, highly contagious disease of young chickens, resulting in immunosuppression and mortality (Toro et al., 2009; van den Berg et al., 2000; Di Fabio et al., 1999). The viral genome codifies for 5 proteins. Viral protein 2 (VP2) is the main structural protein of the capsid, where the neutralizing epitopes are located (Letzel et al., 2007; Lee et al., 2006). The molecular characterization of IBDV can be done by means of mapping the VP2 variable region. So far, sequencing is the most common method used for this type of analysis (Jackwood et al., 2009). Pfizer Poultry Health, provides the poultry sector with global diagnostic services. Amongst the specific tools for IBDV, API (Analysis of digital histological images) is an outstanding methodology to determine the severity of bursal lesions, together with the sequencing of field samples. This work analyses samples submitted during 2010 for characterization by sequencing.
Materials and Methods
During 2010, 187 samples collected from broilers or layers were analyzed at Pfizer Animal Health - Global Poultry laboratory in Durham, NC, to be further submitted to the University of Ohio for IBDV identification. Such samples represent several countries of Latin America and the Caribbean and were collected from birds under field conditions by means of bursal print on FTA cards, suited for the preservation of genetic material and adequate transportation (Moscoso et al., 2006). The variable region of VP2 from all samples was sequenced then analyzed by means of the Lasergene 8 software (DNAStar - MegAlign) (Burland, 2000). The target region includes 234 amino acids covering the VP2 variable region (Lee et al., 2006). Analyses were performed using the Clustal W method then reported by means of phylogenetic trees and identity tables.
Results & Discussion
Of the total amount of 187 IBDV samples isolated from field samples in Latin America in 2010, 86 were RT-PCR positive. All of them were sequenced and analyzed. Samples coming from the same site, an identical sequence appear only once in the phylogenetic tree (Figure 1).
Figure 1. Phylogenetic tree of the Latin-American IBDV isolated in 2010
Most of the samples were characterized as variant strains. Such strains were identified in Colombia, Venezuela, Peru and Jamaica. Very virulent samples were only identified in Colombia. Colombian birds receiving vaccines of the Antigen-Antibody complex type, presented a re-isolated and identified vaccine virus. At present, we are processing samples from farms in Argentina, Brazil, Ecuador. Paraguay and Central America.
Only a few efforts targeting IBDV molecular characterization are performed in Latin America. Most of them are limited to samples collected in one single country. The characterization of 41 samples of Brazilian IBDVs isolated between 1997 and 2005, proved that 60% of them were identified as very virulent (vvIBDV) (Fernandes et al., 2009). In another research, 113 isolates from 18 countries were sequenced, 8 of them were Latin American countries. All samples were from clinical cases that resulted in high mortality rates. Most of the strains were considered as vvIBDV with few variations amongst them (Jackwood & Sommer-Wagner, 2007). Such results differ from the ones found in this study, in which most of the field cases are associated to variant strains. This finding could be an evidence of the increased incidence of variant strains in Latin America. It is necessary to continue performing identification efforts on IBDV strains in order to prove such a trend. This could be beneficial in the control of the disease, because specific vaccination programs could be applied for the particular situations found in each different country.
This study proves the variability of IBDV isolates in Latin America. Most of the samples were identified as variant strains. We will continue with the application of this diagnostic tool, aiming to expand our data base, which will be available for future epidemiological research in Latin America.
Burland TG. 2000. Dnastar's Lasergene sequence analysis software. Methods Mol. Biol. 132:71-91.
Di Fabio J, Rossini LI, Eterradossi N, Toquin MD, Gardin Y. 1999. European-like pathogenic infectious bursal disease viruses in Brazil. Vet. Rec. 145:203-204.
Fernandes, MJ, Simoni IC, Vogel MG, Harakava R, Rivas EB, Oliveira MB, Kanashiro AM, Tessari EN, Gama NM, Arns CW. 2009. Molecular characterization of brazilian infectious bursal disease virus isolated from 1997 to 2005. Avian Dis. 53:449-54.
Jackwood DJ & Sommer-Wagner S. 2007. Genetic characteristics of infectious bursal disease viruses from four continents. Virology. 365:369-75.
Jackwood DJ, Sommer-Wagner SE, Stoute AS, Woolcock PR, Crossley BM, Hietala SK, Charlton BR. 2009. Characteristics of a very virulent infectious bursal disease virus from California. Avian Dis. 53:592-600.
Lee CC, Ko TP, Chou CC, Yoshimura M, Doong SR, Wang MY, Wang AH. 2006. Crystal structure of infectious bursal disease virus vp2 subviral particle at 2.6a resolution: implications in virion assembly and immunogenicity. J. Struct. Biol. 155:74-86.
Letzel T, Coulibaly F, Rey FA, Delmas B, Jagt E, van Loon AA, Mundt E. 2007. Molecular and structural bases for the antigenicity of vp2 of infectious bursal disease virus. J. Virol. 81:12827-12835.
Moscoso H, Alvarado I, Hofacre CL. 2006. Molecular Analysis of infectious bursal disease virus from bursal tissues collected on fta filter paper. Avian Dis. 50:391-396.
Toro H, Effler JC, Hoerr FJ, van Ginkel FW. 2009. Pathogenicity of infectious bursal disease virus variant Al2 in young chickens. Avian Dis. 53:78-82.
van den Berg TP, Eterradossi N, Toquin D, Meulemans G. 2000. Infectious bursal disease (Gumboro disease). Rev. Sci. Tech. 19:509-543.