2005 Poultry Science Association, Inc. Infectious Bronchitis Virus Surveillance in Ontario Commercial Layer Flocks B. Stachowiak,* D. W. Key,† P. Hunton,‡ S. Gillingham,§ and É. Nagy*,1 *Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada, N1G 2W1; †Pennsylvania State University, Animal Diagnostic Laboratory, Orchard Road, University Park, Pennsylvania 16802; ‡Ontario Egg Producers, 7195 Millcreek Drive, Mississauga, Ontario, L5N 4H1, Canada; §Aviagen North America, Cummings Research Park, 5015 Bradford Drive, Huntsville, Alabama 35805 Primary Audience: Egg Producers, Veterinarians, Researchers SUMMARY Infectious bronchitis (IB) is one of the most important viral diseases of poultry and causes major economic losses to the industry. IB status in commercial layers in southern Ontario was assessed through a questionnaire. According to the data gathered from egg producers of Ontario, repeated outbreaks of IB occurred even though flocks had been routinely vaccinated. Based on this information, 13 farms throughout the region were selected for IB virus (IBV) surveillance. IBV vaccinated and unvaccinated sentinel birds were placed in barns with different IB histories, and after a 1-wk exposure tissues and pharyngeal swabs were collected and tested for the presence of IBV. An initial study indicated that tracheal tissues were more often positive than samples from cecal tonsils, kidneys, lungs, and pharyngeal swabs. Therefore, all subsequent IBV detection was directly on tracheal tissues with nucleocapsid (N) gene specific reverse transcription (RT) polymerase chain reaction (PCR). From the sentinel bird placements and N-gene RT-PCR analysis, IBV was detected on 11 farms with differing IB histories. The study indicated that farms with previous IB outbreaks could harbor the virus without clinical signs in the flocks. In addition, some farms, although they did not report having IB associated problems, were IBV positive by N-gene RT-PCR. Key words: infectious bronchitis virus, nucleocapsid gene, sentinel bird, commercial laying hen 2005 J. Appl. Poult. Res. 14:141–146 DESCRIPTION OF PROBLEM Infectious bronchitis (IB) is one of the most important diseases of chickens and continues to cause substantial economic losses to the industry. IB is caused by IB virus (IBV), which is one of the primary agents of respiratory disease in chickens worldwide [1]. All chickens are susceptible to IBV infection, and the respiratory signs include gasping, coughing, rales, and nasal discharge. Sick chicks usually huddle together and appear depressed. The severity of the symptoms in chickens is related to their age and vaccine status. The disease causes some mortality in chicks less than 3 wk of age; in contrast in older birds the clinical 1 To whom correspondence should be addressed: enagy@ovc.uoguelph.ca. Downloaded from https://academic.oup.com/japr/article-abstract/14/1/141/812896 by guest on 29 July 2018 JAPR: Research Report 142 signs may subside quickly. In layers there is a noticeable decline in egg production, egg quality is lowered, and eggs may be deformed with watery albumen. Other signs of IB, such as wet droppings, are due to increased water consumption [2]. The type of virus strain infecting a flock determines the pathogenesis of the disease, in other words the degree and duration of lesions in different organs. The upper respiratory tract is the primary site of infection, but the virus can also replicate in the reproductive, renal, and digestive systems. The outcome of the infection is very much dependent on the immune status of the flock. Chickens that have recovered from IB may have long-term damage in certain organs. For example atrophy of the oviduct contributes to the decline of egg production, which may be up to 50% [3]; poor quality eggshell; and watery albumen. Some IBV strains, such as Aust T that are known for nephropathogenicity, have high tropism to the oviduct epithelium. In addition to commercial layers, eggs from IBVinfected breeder flocks have decreased hatchabilities [4]. After the acute phase of the infection, a persistent infection can be established in specific organs, and the virus can be excreted continuously [5]. Virus shedding can also be stimulated by environmental and physical stresses, leading to a new cycle of infection [6]. Distinctively different serotypes of IBV exist throughout the world. The Massachusetts (Mass) serotype, which was originally identified in the United States, can be found in Europe and Asia, and serotypes different from those in North America have been isolated in other continents [1]. For example, the Australian IB virus strains do not have a close phylogenetic relationship with well-known North American serotypes such as Mass or Connecticut (Conn). However, Australia was the first country to report problems with nephropathogenic IB viruses, which are now recognized as being present in most areas of the world. Differing strains have been identified based on their antigenic variation. Sequence analyses of IBV pathotypes reveal that they are distinct in each geographic region. In general, the incidence of IB outbreaks is sporadic and predominantly depends on the vaccination practices specific to each country, regardless of the virus pathotype [6, 7]. Sentinel chickens raised for viral disease surveillance have been employed successfully to investigate a variety of viruses such as West Nile virus, infectious bursal disease virus and IBV [8, 9, 10, 11]. Because sentinels are highly susceptible to infection, the likelihood that they will contract the infectious disease agent when placed on suspect farms is increased. The use of sentinel birds exposed to flocks with persistent viral infections could increase the chances of virus recovery and reduce false negative diagnosis [12]. Broiler and layer flocks have been monitored for the presence of IBV by sentinel birds [10, 12]. Sentinel birds have also been used to investigate persistent IB problems on farms for the purpose of virus reisolation [10, 11] and to demonstrate that IBV-vaccinated sentinel birds could be used to evaluate the efficacy of vaccines [12]. A prestudy survey was conducted among egg producers in Ontario to assess the number of farms affected by IB. A few producers (13%) reported continuous problems associated with IB, whereas 87% of the responders did not indicate any IB problems. Based on the responses to the questionnaire, the prevalence of IB was 14.2%. The aims of this study were to survey the status of IBV through sentinel birds in Ontario layer operations and to adapt and evaluate a method to detect IBV rapidly in tissues. MATERIALS AND METHODS Sentinel Birds One-day-old Shaver Leghorn chickens [13] were obtained and housed in the isolation unit of the University of Guelph as outlined in the Animal Utilization Protocol. The birds were vaccinated against Newcastle disease virus, and half of them were vaccinated against IB [14] by an eye drop method at 3 wk of age. At 6 wk of age, 6 vaccinated and 6 unvaccinated chickens were transported to selected farms. In the barns the caged chickens were placed in close proximity to the flock and in direct airflow of ventilation units. After 1 wk of exposure, the sentinel birds were transported back to the laboratory where they were euthanized, and samples of organs (lung, kidney, and cecal tonsils), swabs (pharyngeal and cloacal), and blood were collected. The Downloaded from https://academic.oup.com/japr/article-abstract/14/1/141/812896 by guest on 29 July 2018 STACHOWIAK ET AL.: INFECTIOUS BRONCHITIS VIRUS SURVEILLANCE 143 TABLE 1. Description of facilities and infectious bronchitis (IB) history of selected farms N-gene specific RT-PCR results1 Farm Flock size Flock age A B-13 B-23 B-33 C 7,000 16,100 49,698 48,385 18,600 Multiple age 32 wk 32 wk 32 wk 38 wk D E F G H M Q R S X 12,000 18,200 3,400 2,672 7,400 34,000 19,250 11,178 13,121 59,600 59 wk 44 wk NA5 20 wk 35 wk 40 wk 34 wk 32 wk 35 wk 60 wk Clinical IB Status2 Unvaccinated sentinels Vaccinated sentinels Total Respiratory signs Egg color discoloration None4 None <1 yr 6/6 3/6 4/6 6/6 5/6 4/6 2/6 3/6 3/6 4/5 10/12 5/12 7/12 9/12 9/11 2 mo 3 mo None 8 mo <1 yr None None <1 yr <2 yr None 4/6 2/6 0/6 6/6 4/6 4/6 1/6 0/2 0/6 4/6 49/86 0/5 0/6 0/6 4/6 4/6 4/6 0/6 0/6 1/5 4/6 33/87 4/11 2/12 0/12 10/12 8/12 8/12 1/12 0/8 1/11 8/12 82/173 Breed White Leghorns White Leghorns White Leghorns Bovans Bovans Browns Dekalb Delta Babcock White Leghorns Dekalb Delta White Leghorns White Leghorns ISA brown Babcock White Bovans Hyline 98 1 Results of IB virus detection with N-gene specific reverse transcription-polymerase chain reaction (RT-PCR) in tracheal tissues from sentinel birds. Clinical IB status prior to sentinel bird placement. 3 The number indicates different barns on the same farm. 4 None = the farm did not indicate any problems associated with IB. 5 Not available. 2 flow chart of the experimental design is shown in Figure 1. practices and IB history. Some of the data gathered are summarized in Table 1. Farm Selection for Sentinel Bird Placement N-Gene Specific RT-PCR to Detect IBV in Tissues Thirteen farms participated in the study (Table 1). Some of the farms (A, C, D, E, M, Q) were identified based on information obtained from veterinary clinicians, who were aware of the IB problems. Other farms (B, F, G, H, R, S, and X) were selected based on the survey described in the introduction. These farms were located in one county of Ontario, where higher incidences of IB-related problems were identified. The potential farms chosen for sentinel placement had to have permission from the Ontario Egg Producers to be included in the study. If a given producer did not wish to participate in the study, the producer at the farm with the next most recent IB outbreak was contacted. At the time of the bird delivery, a detailed survey was given to the producer to be filled out and returned when birds were collected. The survey asked for information regarding flock size, breed, vaccination programs, farm management Total RNA was extracted from approximately 100 mg of tissue and homogenized in 1 mL of TRIzol [15] and kept at room temperature for 5 min. After the addition of 240 µL of chloroform it was shaken vigorously and incubated for 10 min. The sample was centrifuged and the upper colorless aqueous phase was transferred to a clean tube and the RNA was precipitated by addition of an equal volume of isopropyl alcohol. The RNA pellet was dissolved in 10 µL of Rnase-free water and stored at −70°C. To detect all serotypes of IBV a conserved region of the genome such as the nucleocapsid (N) gene was targeted by reverse transcription (RT) polymerase chain reaction (PCR). The primer pair used in this study was published by Handberg et al. [16], and the 2-step method was followed as described [17]. Briefly, the random hexamer primed cDNA synthesis was carried Downloaded from https://academic.oup.com/japr/article-abstract/14/1/141/812896 by guest on 29 July 2018 JAPR: Research Report 144 FIGURE 1. Flow chart of the experimental design. Farm selection was based on a questionnaire directed to all commercial layer operations in Ontario. Of the 12 sentinel birds, half of birds were vaccinated against infectious bronchitis virus (IBV), and the other half was not vaccinated. out with Superscript II RNase H reverse transcriptase. The PCR reactions were conducted in a GeneAmp Applied Biosystems 9600 thermocycler using Invitrogen reagents [15, 18]. The PCR products were analyzed in 0.8% agarose gels prepared in 1× TAE (40 mM Tris, 5 mM sodium acetate, and 1 mM EDTA, pH 7.8). The DNA bands were visualized by staining with 0.2 mg/mL of ethidium bromide and viewing on an ultraviolet transilluminator at 366 nm wavelength using a BioRad Gel Doc 1000 system [19]. RESULTS AND DISCUSSION Three hundred sentinel chickens were raised, and 1,800 samples were collected during this study. Tissues and pharyngeal swabs were collected from each sentinel bird for testing to determine whether the sentinel birds had become infected with IBV during the farm placement. FIGURE 2. Agarose gel electrophoresis of reverse transcription-polymerase chain reaction products using infectious bronchitis virus (IBV) N-gene specific primers and RNA extracted from tracheal tissues of sentinel birds placed on farm B-1. Lanes 1–6: different sentinel birds, lane 7: known positive sample, lane 8: IBV Mass 42, lane 9: negative control trachea, lane M: 100-bp ladder. Diagnostic laboratories usually first isolate the virus in embryonated eggs and use the allantoic fluid to detect IBV specific RNA by RTPCR. To reduce the time and labor needed for diagnosis, we first assessed the feasibility of the N-gene specific RT-PCR to detect virus directly in tissues without virus isolation. Trachea, lung, kidney, and cecal tonsils from birds placed on 3 farms (A, Q, E) were analyzed. Representative RT-PCR results are shown in Figure 2. A sample was considered positive when the expected size fragment (450 bp) was observed in the gel. As expected, IB virus was detected more frequently in tracheal tissues than in lungs, kidney, or cecal tonsils. Overall 62.5% of the IBV-positive tissues were from farm A, and only 2 (8.3%) and 3 (12.5%) originated from farms E and Q, respectively (Table 2). In a study carried out by Handberg et al. [16] IBV was detected in the tracheas of experimentally inoculated chickens. In this study we were able to detect the virus in TABLE 2. N-gene specific reverse transcription-polymerase chain reaction results for RNA extracted from different tissues for detection of infectious bronchitis virus (IBV) Farm A Q E Total 1 Trachea 1 6/6 2/6 2/6 10/18 Lung Kidney Cecal tonsils Total IBV positive 3/6 1/6 0/6 4/18 2/6 0/6 0/6 2/18 4/6 0/6 0/6 4/18 15/24 3/24 2/24 20/72 62.5% 12.5% 8.3% Number of positive per number of birds. Downloaded from https://academic.oup.com/japr/article-abstract/14/1/141/812896 by guest on 29 July 2018 STACHOWIAK ET AL.: INFECTIOUS BRONCHITIS VIRUS SURVEILLANCE TABLE 3. Statistical analysis of virus detection by Ngene specific reverse transcription-polymerase chain reaction (RT-PCR) done directly on tissues versus infectious bronchitis virus (IBV) isolation followed by RT-PCR evaluated by a 2 × 2 table1 Tissues +2 Tissues −3 Allantoic fluid +2 Allantoic fluid −3 Total 22 3 25 0 5 5 22 8 30 1 Sensitivity: 88% (confidence interval: 67.7 to 96.8%); specificity: 100% (confidence interval: 46.3 to 100%). 2 + = positive IBV detection. 3 − = negative IBV detection. field-exposed birds. Although the trachea is the preferred tissue for virus detection in the case of acute infection, in subclinically affected flocks, kidney and cecal tonsils might be a better choice when testing for persistent infection [5, 20]. The sensitivity and specificity of direct virus detection in tissues by RT-PCR were compared to virus isolation followed by RT-PCR. The tracheal tissues were randomly selected from 30 sentinel birds representing farms A, D, E, M, and Q. For virus isolation samples were passaged in embryonated specific pathogen-free eggs 3 times or until stunting or curling of the embryos was observed, then RNA was extracted from the allantoic fluid and subjected to N-gene specific RT-PCR. The results and statistical analysis of the 2 approaches are summarized in Table 3. Sensitivity and specificity percentages were determined using the EPI Info 6 program [21]. Direct virus detection in tissues was 100% specific and 88% sensitive when compared with virus isolation followed by RT-PCR. These results were considered satisfactory to proceed with the larger study. Of the 402 egg producers throughout southern Ontario, 13 farms participated in the sentinel bird study. The selected farms were at least 25 km apart from each other and were not centered in one area. Of the surveyed farms, 11 were positive by the N-gene specific RT-PCR when tissues of unvaccinated and vaccinated sentinel birds were tested after a 1-wk exposure. The vaccinated chickens were used to evaluate the protection afforded by the most commonly used vaccination programs for Ontario broilers, lay- 145 ers, and breeders. Overall, IBV was more often detected in tracheal tissues of unvaccinated than vaccinated birds (Table 1). It seemed that the mild IBV vaccine did not protect the sentinel chickens against infection. If the unvaccinated chickens became infected so did the vaccinated birds, regardless of the IB history of the farm. The antibody (Ab) level of vaccinated birds may not have been high enough to protect the chickens from infection. Based on the data of de Herdt et al. [22], the Ab titers of vaccinated sentinel chickens in this study were considered relatively low, partly because of the single vaccination. Moreover, Gelb et al. [12] demonstrated that ocular vaccination with the Mass serotype alone does not prevent IBV infection. However, vaccination with the Mass serotype could reduce the rate of mortality because this serotype provides some heterologous protection against other IBV strains [23]. Indeed, according to this study, the clinical signs were less severe in vaccinated birds than in unvaccinated birds placed on farm A. Clinical signs of IB were observed at the time of sentinel bird placement in this layer flock. The IBV infection is highly contagious, and the virus is easily transmitted [2]. Indeed the sentinel birds exposed to the layer flocks (farms A and B-1) with clinical signs contracted the virus. Also, farms with outbreaks less than 3 mo (farms D and E) prior to sentinel bird placement were positive by N-gene specific RT-PCR. IBV can establish a persistent infection, and the virus can be isolated from flocks up to 39 d postinfection [5]. Moreover, IBV can survive in fecal material [1], permitting infection of susceptible birds. Farms with a history of IB outbreaks could harbor the virus without exhibiting clinical signs of the disease or presenting problems as was shown by our study. Farms B-2, B-3, M, and X did not report problems associated with IB, yet IBV was detected on these farms by the N-gene specific RT-PCR. Detection of IBV with N-gene specific RTPCR cannot differentiate a vaccine strain from field virus isolates. Therefore, the next step will be to partially characterize these viruses by using an S1-gene specific primer in the PCR reaction followed by restriction fragment length polymorphism and sequence analyses [17]. Downloaded from https://academic.oup.com/japr/article-abstract/14/1/141/812896 by guest on 29 July 2018 JAPR: Research Report 146 CONCLUSIONS AND APPLICATIONS 1. The main advantage of IBV detection by direct tissue extraction was increased speed of obtaining results as opposed to virus isolation from eggs. The detection of IBV could be reduced from weeks to 1 d. 2. The cDNA generated for N gene specific PCR could be used in the PCR with S1-gene specific primers so the IBV isolate can be quickly characterized. Nevertheless, virus isolation from specific pathogen-free eggs cannot be eliminated. Infectious virus may be necessary for additional tests, for example, to evaluate the efficacy of vaccine protection or to assess the serotype of the isolate. The combination of the 2 virus detection systems could be the optimal diagnostic tool. 3. Use of vaccinated and unvaccinated sentinel birds was shown to be highly effective as a means of identifying IBV-positive barns regardless of whether they had a history of IB-related signs. 4. Knowledge of the persistence of the virus in these barns in the absence of detectable clinical signs is of great importance to the development of effective long-term vaccination programs in individual barns and in multiple-barn complexes. REFERENCES AND NOTES 1. Cavanagh, D., and S. A. Naqi. 2003. Infectious Bronchitis. 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Acknowledgment This work was supported by the Ontario Egg Producers, the Ontario Ministry of Agriculture and Food, and the Poultry Industry Council, Ontario, Canada. We thank Sophie St-Hilaire (Department of Population Medicine, University of Guelph) for her advice on statistical analysis and Helena Grgic (Department of Pathobiology, University of Guelph), and David Bridle (Isolation Unit, University of Guelph) for their assistance in the experimental work. Downloaded from https://academic.oup.com/japr/article-abstract/14/1/141/812896 by guest on 29 July 2018