Risk factors and characteristics of low pathogenic avian influenza virus isolated from commercial poultry in Tunisia

Abstract

Objective: Estimate the seroprevalence of influenza A virus in various commercial poultry farms and evaluate specific risk factors as well as analyze their genetic nature using molecular assays.

Materials and methods: This report summarizes the findings of a national survey realized from October 2010 to May 2011 on 800 flocks in 20 governorates. Serum samples were screened for the presence of specific influenza virus antibodies using cELISA test. Additionally, swab samples were tested by real time and conventional RT-PCR and compared with results obtained by others assays. Phylogenetic and genetic analyses of the glycoproteins were established for some strains.

Results: Out of the 800 chicken and turkey flocks tested by cELISA, 223 showed positive anti-NP antibodies (28.7%, 95% CI: 25.6-32.1). Significantly higher seroprevalence was found among the coastal areas compared to inland and during the autumn and winter. Broiler flocks showed significantly lower seroprevalence than layers and broiler breeders. The influenza virus infection prevalence increased after the laying phase among layer flocks. In addition, AIV seropositivity was significantly associated with low biosecurity measures. The Ag EIA and rRT-PCR tests revealed significantly higher numbers of AI positive samples as compared to cell cultures or egg inoculation. All new strains were subtyped as H9N2 by real time and conventional RT-PCR. Drift mutations, addition or deletion of glycosylation sites were likely to have occurred in the HA and NA glycoproteins of Tunisian strains resulting in multiple new amino acid substitutions. This fact may reflect different evolutionary pressures affecting these glycoproteins. The role of these newly detected substitutions should be tested.

Conclusion: Our findings highlight the potential risk of AIV to avian health. Strict enforcement of biosecurity measures and possible vaccination of all poultry flocks with continuous monitoring of poultry stations may ensure reduction of AIV prevalence and avoid emergence of more pathogenic strains.

Figure 1. cELISA results for detection of antibodies to influenza A virus NP in commercial chicken and turkey sera.

(Result was defined as positive when % of competition is ≤45%, doubtful at 45–50% and negative ≥50% competition).

Figure 2. The monthly distribution of AIV between-flock seroprevalence (%) in commercial poultry flocks Tunisia (778 chicken and turkey flocks sampled from October 2010 to May 2011).

Figure 3. The between-flock AIV seroprevalence (mean proportion of poultry flocks positive regarding antibodies to AIV) in Tunisian governorates from October 2010 to May 2011.

Figure 4. Map of the estimated between-flock AIV seroprevalence and number of commercial poultry flocks sampled by Tunisian governorates from October 2010 to May 2011.

Figure 5. Pathogenicity of CEF cells to avian H9N2 influenza virus in the presence (CDEF) and absence (B) of trypsin.

(A) mock cells. Arrows showed syncytia and plaque formations.

Figure 6. A linear relationship between threshold cycle and serially diluted DNA concentration or TCID50 values.

PCR efficiency ((10^3.503−1)×100) was 99.87% as indicated by the slope (m = −3.503). The standard curve was generated from amplification of the H9 gene with each point represents the mean of the results from three determinations.

Figure 7. Phylogenetic relationships of the HA (a) and NA (b) genes of 2010–2011 Tunisian strains (H9N2).

Horizontal distances are proportional to the minimum number of nucleotide differences required to join nodes and sequences. Vertical distances are for spacing branches and labels. The phylogenetic trees were generated by using the neighbor-joining algorithm assessed by bootstrap analysis with 1,000 replications. Abbreviations used in virus designation are as follows: Av, avian; Ck, chicken; Dk, duck; gs, goose; Qu, quail; Tk, Turkey; Pa, parakeet. The in box strains are avian H9N2 Influenza viruses' isolates that were sequenced in the present study.