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Brief Research Reports |
Correspondence: 1Corresponding Author: Claudia Muñoz-Zanzi, 1300 S Second Street, Suite 300, Minneapolis, MN 55454, e-mail: munozzan{at}umn.edu
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Key Words: Avian metapneumovirus • diagnostics • enzyme-linked immunosorbent assay • specificity • turkeys
Avian metapneumovirus (AMPV; order Mononegavirales; family Paramyxoviridae; subfamily Pneumovirinae; genus Metapneumovirus)11 is an RNA virus that was first identified in the United States in 1996 from a commercial turkey flock in Colorado following an outbreak of upper respiratory disease.14 In 1997, the virus was detected in Minnesota, where it became endemic in subsequent years8 with a flock seroprevalence ranging from 14% to 65%.9 The virus was later detected in other states, including North Dakota, South Dakota, and Wisconsin.2,13 The incidence of AMPV outbreaks in domestic turkeys in Minnesota has shown a seasonal trend, with most outbreaks occurring in spring and fall. It has been speculated that this pattern could be explained by migrations of wild birds, which may serve as reservoirs for AMPV.2 Impact of AMPV infection on flock health includes decreased egg production, morbidity up to 100% in flocks showing clinical disease, and mortality that can be a low as 0.4% or as high as 50%.7
Routine serologic testing for AMPV infection of turkey flocks at slaughter is currently being used to monitor changes in the occurrence of AMPV infection in endemic areas and can also be used to detect the emergence of infection in currently unaffected areas. The goal of a monitoring program at slaughter is to identify infected flocks through detection of AMPV antibodies. However, serologic assays are not perfect, and false-positive and false-negative results are expected. Consequently, knowledge of the accuracy of the diagnostic test is needed to interpret the observed results appropriately. Avian metapneumovirus serologic testing is commonly done using an enzyme-linked immunosorbent assay (ELISA), which has been evaluated to some extent. Accuracy evaluations have been performed using samples from individual birds under experimental infection settings. Reported estimates of diagnostic sensitivity (SE) for ELISA include 98%12 and 99%,10 and estimates of 98%12 and 99.5%10 for diagnostic specificity (SP). Because the interest of a monitoring program is to identify infected flocks as accurately as possible, and not necessarily individual infected birds, the parameters of diagnostic performance needed are flock-level SE and SP. Flock-level SE is defined as the probability that an infected flock is classified as infected, and flock-level SP is defined as the probability that an uninfected flocks is classified as uninfected. Because of the costs associated with false-positive results, particularly in areas that are free of AMPV infection, there is a need to obtain improved estimates of flock-level SP. The objective of the current study was to estimate flock-level SP of a program to monitor AMPV infection in turkeys at processing using a standard ELISA and to describe possible factors associated with false-positive results.
The objective was accomplished through a prospective study of AMPV-free flocks, which were tested for AMPV antibodies at various times. Turkey producers from a geographic area of Iowa known to be free of AMPV, based on historical evidence of no infection, were invited to participate in the study. A total of 7 farms were enrolled in the study based on their willingness to collaborate by enrolling flocks in the prospective study. Farms can be considered representative of independent, family-owned turkey production facilities in the Midwest. The target sample size for number of flocks was based on an expected value for flock-level SP of 80%, 95% confidence level, margin of error of 20%, and a 50% increase beyond the recommended sample size to allow further statistical analysis. A questionnaire was administered to the farm owner to request information associated with each flock enrolled in the prospective study, including hatch date, placement date, flock size, mortality, vaccinations, treatments, clinical disease, sampling dates, and processing date.
Blood samples were obtained by venipuncture in collaboration with the farm owner at 8 weeks and 12 weeks of age and immediately before processing for slaughter. At each sampling, 20 birds were randomly selected from each flock and bled to obtain 2 ml of blood, which was sent to the Iowa Veterinary Diagnostic Laboratory (Ames, IA) for processing. Sera were stored at –20°C until testing at the Minnesota Poultry Testing Laboratory (Willmar, MN). The current AMPV surveillance protocol in Minnesota involves sampling10 birds per flock; however, based on the known negative correlation between sample size and population-level SP,5 the number of birds per flock was doubled to obtain a more conservative estimate of flock-level SP.
Samples were tested for AMPV using the same ELISA procedure that is used as part of the routine AMPV monitoring program in Minnesota.4 Briefly, serum samples were diluted 1:40 in dilution:blocking buffer and added to 2 virus antigen-coated wells (antigen prepared in Vero [African green monkey kidney] cells) and 2 control antigen-coated wells (equally processed uninfected Vero cells) at 50 µl/well. Known positive and negative control sera were included in each plate. The plates were covered, incubated at room temperature for 1 hr, washed 5 times, and blotted dry. The conjugate was diluted in diluting-blocking buffer and added to each well at 50 µl/well. After incubation at room temperature for 1 hr, the conjugate was removed, and the plates were washed 5 times and blotted dry. Substrate solution was added to each well at 100 µl/well. After incubation in the dark for 10 min, the reaction was stopped by adding 25 µl of 2.5 mol sulfuric acid to each well, and the optical density (OD) was read at 490/405 nm. The results were expressed as the average OD difference (ODD) between virus antigen-coated and control wells of each serum. A sample with ODD of 
0.2 was considered positive. Additionally, samples were tested for Newcastle disease virus (NDV), Avian influenza virus (AIV), and Bordetella avium, as a predetermined respiratory disease panel using commercially available ELISA diagnostic test kits.a
Descriptive statistics (means, proportions, and standard deviations) were used to characterize the management and sanitary conditions of the farms and flocks enrolled in this study. Traditionally, monitoring programs assume that a flock is infected if at least 1 sample tested positive; however, sometimes this flock-level cutoff is arbitrarily increased because of concern with false-positive results. For a given sampling, flocks in the current study were classified based on AMPV infection status using flock-level cutoffs from 1 through 4 positive samples. Flock-level, diagnostic SP for a monitoring program based on testing 20 birds per flock was defined as the proportion of these AMPV-free flocks that were correctly classified as AMPV negative. To account for the correlation of flocks within farms, population-averaged estimates of flock-level SP and 95% confidence intervals (CI) were calculated using a generalized estimating equation model for binomial data and a exchangeable correlation structure.1 The same model was used to assess the potential effect of flock factors, including flock size, mortality, clinical disease, vaccination, treatments, and serostatus for other infections on the probability of (false) positive AMPV results. The assumption of linearity in the ln(odds) for continuous variables (mortality proportion, proportion positive for NDV, AIV, and B. avium) was assessed by categorizing each variable and visually inspecting a plot of the estimates of ln(odds) against the midpoint of the categories.6 Because of limited sample size for the statistical analysis, a P-value of <0.1 was considered statistically significant. All statistical analysis was performed using available computer packages.b,c
A total of 37 flocks from 7 different farms were followed prospectively from 8 weeks of age until processing at approximately 21 weeks of age. Between 1 and 13 flocks per farm were enrolled. Avian metapneumovirus test results were available for 732 birds at 8 weeks of age, 721 birds at 12 weeks of age, and 465 birds at processing. Questionnaire information was available for 28 flocks, which was used to describe the characteristics of the farms and flocks (Table 1). The majority of the flocks had 10,000 birds and were administered routine vaccinations for B. avium and Hemorrhagic enteritis virus. Observation of the distributions of the bird-level crude ODD values showed a shift to the left as age increased (Fig. 1) and, therefore, the number of samples that yielded a positive AMPV ELISA (ODD 0.2) decreased with time. A total of 42 of 732 (6%) samples tested AMPV positive at the first sampling, 20 of 721 (3%) at the second sampling, and 1 of 465 (0.2%) at processing.
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An interesting finding of the present study was that the likelihood of (false) positive results decreased as age increased (Table 2). This could be explained by potential cross-reactions with maternal immunity, which wanes gradually over time; however, it commonly disappears by 4 weeks after hatch.3 Statistical analysis of the factors associated with the probability of a (false) AMPV-positive flock was performed with data from the first sampling and a cutoff of 1. Results suggested that administration of antibiotics any time before the first sampling (P = 0.02) and vaccination for B. avium <14 days before the first sampling (P = 0.08) were positively associated with the probability of (false) positive test results. These associations are indicative of possible cross-reactions of the AMPV test with immune responses to the vaccines and/or other infections that were not examined in this study. The fact that 77% of the vaccinations or treatments occurred before the first sampling may help explain the decrease over time in the number of false positive results that was observed. The potential effect of cross-reactions highlights the need to consider variable performance of diagnostic depending on farm conditions. Overall, findings from this study suggest that monitoring for AMPV at processing is highly specific; however, estimates of SP cannot be extrapolated to other poultry farming systems, such as backyard flocks, or large, vertically integrated commercial farms, or farms from different geographical areas.
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Synbiotics Corporation, San Diego, CA.
R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria.
Excel® 2007, Microsoft Corporation, Seattle, WA.
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