Optimal specimen collection and transport methods for the detection of avian influenza virus and Newcastle disease virus

Abstract

Background: Active and passive surveillance for avian influenza virus (AIV) and Newcastle disease virus (NDV) is widespread in commercial poultry worldwide, therefore optimization of sample collection and transport would be valuable to achieve the best sensitivity and specificity possible, and to develop the most accurate and efficient testing programs. A H7N2 low pathogenicity (LP) AIV strain was selected and used as an indicator virus because it is present in lower concentrations in swabbings and thus requires greater sensitivity for detection compared to highly pathogenic (HP) AIV. For similar reasons a mesogenic strain of NDV was selected. Using oro-pharyngeal and cloacal swabs collected from chickens experimentally exposed to the viruses we evaluated the effects of numerous aspects of sample collection and transport: 1) swab construction material (flocked nylon, non-flocked Dacron, or urethane foam), 2) transport media (brain heart infusion broth [BHI] or phosphate buffered saline [PBS]), 3) media volume (2 ml or 3.5 ml), 4) transporting the swab wet in the vial or removing the swab prior to transport, or transporting the swab dry with no media, and 5) single swabs versus pooling 5 or 11 swabs per vial.

Results: Using real-time RT-PCR (rRT-PCR), virus isolation (VI) and commercial antigen detection immunoassays for AIV we observed statistically significant differences and consistent trends with some elements of sample collection and transport; media, dry transport and swab construction. Conversely, the number of swabs pooled (1, 5 or 11) and whether the swab was removed prior to transport did not impact virus detection. Similarly, with NDV detection by both VI and rRT-PCR was not affected by the numbers of swabs collected in a single vial (1, 5 or 11).

Conclusions: We observed that flocked and foam swabs were superior to non-flocked swabs, BHI media was better than PBS, and transporting swabs wet was better for virus recovery and detection than transporting them dry. There was no observable difference in detection whether the swab was removed prior to transport or left in the vial. Also, with both AIV and NDV, there was no observed difference in virus detection between pools of 1, 5 or 11 swabs.

Figure 1

Mean AIV titers by quantitative real-time RT-PCR for each swab construction type by day post inoculation and transport media: a) brain heart infusion (BHI), b) phosphate buffered saline (PBS). Statistical significance (p ≤ 0.05) in differences among the amount of virus recovered by swab construction type for each day post inoculation is indicated by different letters above the bars. Error bars represent standard deviation.

Figure 2

Mean AIV titers by quantitative real-time RT-PCR for each transport condition. Statistical significance (p ≤ 0.05) in differences among the amount of virus recovered by transport condition for each day post inoculation is indicated by different letters above the bars. Error bars represent standard deviation.

Figure 3

Mean AIV titers by quantitative real-time RT-PCR for a single swab or pools of 5 or 11 swabs. Statistical significance (p ≤ 0.05) in differences among the amount of virus recovered by numbers of swabs pooled for each day post inoculation is indicated by different letters above the bars. Error bars represent standard deviation.

Figure 4

Swab types; a) non-flocked Dacron, b) nylon flocked, c) urethane foam, d) culturette.

Figure 5

Diagram of experiment to evaluate swab type and media type. Specific pathogen free white leghorn chickens were inoculated with 10^6.5 50% egg infectious doses of AIV by in the intrachoanal route. Fifteen oro-pharyngeal and 15 cloacal swabs were collected at 1, 2, 3, 4, 7, 10, 14, 17 and 21 days post inoculation using non-flocked swabs, flocked swabs and foam swabs. Individual swabs for each swab construction type were transported in either brain heart infusion (BHI) broth or phosphate buffered saline (PBS).

Figure 6

Diagram of experiment to evaluate transport conditions and media volume. Specific pathogen free white leghorn chickens were inoculated with 10^6.5 50% egg infectious doses of AIV by in the intrachoanal route. Oro-pharyngeal (OP) and cloacal (CL) swabs were collected at 1, 2, 3, 4, 7, 10 and 14 days post inoculation using flocked swabs and brain heart infusion broth (BHI). Individual swabs and pools of 5 swabs (4 from unexposed chickens and 1 from an inoculated chicken) were collected and trans ported in different conditions as follows: 1) collected in 3.5 ml of BHI and transported either in the swab tube n = 15 OP and 15 CL swabs; 2) collected in 3.5 ml BHI and wrung out in the swab tube and removed prior to transport; 3) collected in 2 ml BHI and wrung out in the swab tube and removed prior to transport, n = 10 OP and CL swabs each (individual swabs only); 4) collected in 2 ml of BHI and transported in the swab tube, n = 10 OP and CL swabs each (only individual swabs collected); 5) collected and transported dry, pooled and transferred to BHI 24 hours after transport, n = 15 OP and 15 CL swabs each.

Figure 7

Diagram of experiment to evaluate swab pooling. Specific pathogen free white leghorn chickens were inoculated with 10^6.5 50% egg infectious doses by in the intrachoanal route. Oro-pharyngeal (OP) and cloacal (CL) swabs were collected at 1, 2, 3, 4, 7 and 14 days post inoculation using non-flocked swabs and brain heart infusion broth. Twenty (2 replicates of 10) individual OP or CL swabs were collected at each time point. Thirty OP and 30 CL (2 replicates of 15) of each of the 5 and 11 swab pools were collected at each time point. Pools were comprised of 1 swab from a chicken inoculated with LPAIV and the remaining (4 or 10) swabs were collected from unexposed chickens.