Super-sentinel chickens and detection of low-pathogenicity influenza virus.

Chicken interferon-[alpha] administered perorally in drinking water acts on the ompharyngeal mucosal system as an adjuvant that causes chickens to rapidly seroconvert after natural infection by low-pathogenicity Influenza virus. These chickens, termed super sentinels, can serve as sensitive early detectors of clinically inapparent infections.

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Early detection of low-pathogenicity type A influenza virus (LPAI) circulating among chickens is important for 3 reasons: 1) these are the most prevalent strains in nature and can cause substantial losses for commercial poultry producers (1), 2) these strains can contribute genetic material to high-pathogenicity type A influenza virus (HPAI) (2), and 3) the H5 and H7 LPAI strains can mutate to HPAI with catastrophic effects in birds and with the potential for transmission to humans with lethal consequences (3). Kuiken et al. reported that an HPAI (H7N7) isolate was observed in February 2003 in the Netherlands, which most likely originated in free-living ducks and had evolved into a highly pathogenic variant after introduction into poultry farms (4). Although subsequent serologic screening of poultry showed that the H7 influenza virus had been affecting the Dutch poultry industry several months before the major epidemic, its presence had not been recognized (4). Our study addresses this problem by using a novel method that causes chickens to seroconvert under conditions in which LPAI would otherwise go undetected. This report shows that recombinant chicken interferon-[alpha] (rChIFN-[alpha]) (5) administered perorally in drinking water (6) acts as an adjuvant to produce a super-sentinel chicken that is a sensitive and early detector of clinically inapparent LPAI.

The Study

In 2003, the first clue to an aberrant condition in a commercial flock of laying hens in Connecticut was signaled by a drop in feed consumption and then in egg production. It took 6-7 weeks from the time tracheal samples were sent to a diagnostic laboratory to confirm the diagnosis of LPAI (H7N2) infection at National Veterinary Services Laboratory (NVSL) (N. Adriatico, pers. comm.). One such isolate, A/CK/CT/72/2003(H7N2), was obtained from the US Department of Agriculture, NVSL, Ames, Iowa, and used throughout this study to determine whether the peroral administration of rChIFN-[alpha] under conditions found to ameliorate Newcastle disease (6), infectious bronchitis (7), and infectious bursal disease (8), would similarly affect avian influenza. We reasoned that if the spread of LPAI could be slowed or prevented, the probability of its mutating to HPAI would be proportionately reduced, thereby lowering the chances of transmission to humans. In the course of this study, we observed a strong adjuvant effect of rChIFN-[alpha] administered in drinking water under conditions of virus transmission that mimic natural infection in chickens. This led to the concept of the super-sentinel chicken described here.

Three-week-old specific-pathogen-free (SPF) white leghorns (Charles River Specific Pathogen Free Avian Supplies [SPFAS], Inc., Storrs, CT, USA) were tagged and divided into 2 groups of 10 chickens each. Two birds in each group were overtly infected intravenously or intranasally with [10.sup.6] infectious particles, measured as plaque-forming particles in primary chicken kidney cells (Charles River SPAFAS, Inc.). This strain of LPAI (H7N2) required a high inoculum to ensure infection (data not shown), comparable to that reported for another LPAI (H7N2) strain evaluated in SPF chickens (9). The 8 remaining cage mates in each group served as sentinel birds naturally subject to infection by the respiratory tract, ingestion of fecal material, or both. One group of birds received plain drinking water; the other group received drinking water that contained 2,000 U/mL rChIFN-[alpha]. The water was provided ad libitum and changed daily. Water consumption was the same in both groups, as determined from the amount remaining after a known volume was provided each day (data not shown). With a half-life of 3-5 days in water at room temperature (6), this concentration of interferon (IFN) delivered an average dose of [approximately equal to] 3 x [10.sup.5] U rChIFN-[alpha]/bird/day. Fourteen days post overt infection (dpi), the rChIFN-[alpha]-water was replaced with plain water for the remaining 14 days of the study. This dose of rChIFN-[alpha] was sufficient to ameliorate Newcastle disease (6).

Following overt infection of 2 birds per cage, and the natural cross-infection of the 8 cage mates, serum samples were taken from each of the 10 birds at the intervals indicated in Figure 1. This figure shows data from 2 independent studies that used agar gel precipitin (AGP) tests to detect antibody against avian influenza virus nucleoprotein and M1 antigens. This qualitative test demonstrated that of the 16 naturally infected chickens given plain water, none seroconverted over the 28-day period they were exposed to the 2 infected cage mates. In marked contrast, of the 16 naturally infected chickens given water containing IFN, 14 were seropositive by 14 dpi and remained so during the 28-day test period.

[FIGURE 1 OMITTED]

Figure 2 shows the number of seroconverted birds in a third study as quantified by hemagglutination inhibition (HI) titer (HI U/mL) of serum samples taken at the time intervals indicated as dpi. None of the 8 naturally infected birds given plain water seroconverted during the 28 days of the trial. In contrast, the 8 naturally infected chickens raised on IFN-water all seroconverted by 10 dpi (8/8), as did the overtly infected birds. Similar results were observed in 2 other trials. In all, 4 independent comparable trials were conducted, representing 2 AGP and 2 HI tests (Table 1). The marked contrast in the fraction of naturally infected birds that seroconverted on plain water and IFN-water is evident.

Although the sensitivity of LPAI to the action of IFN is well documented (10,11), rChIFN-[alpha] in the drinking water may have been exacerbating the infection, thereby leading to high levels of virus and antigen and high levels of seroconversion. This possibility was tested by using quantitative real-time reverse transcriptase-PCR to determine the amount of avian influenza virus in tracheal samples at 2, 4, and 10 dpi. Table 2 shows that within the error expected from testing individual chickens, the amount of infectious particle equivalents were not significantly different in birds given plain water or IFN-water. Thus, that more avian influenza virus antigen was produced in chickens that were given IFN-water is an unlikely explanation.

[FIGURE 2 OMITTED]

Conclusions

Although the role of IFN as an adjuvant when delivered perorally has been established in mammals (12), our data demonstrate for the first time, to our knowledge, that avian IFN administered in drinking water to naturally infected chickens lowers the threshold of antigen required to stimulate the adaptive immune response to an LPAI isolate. As a consequence, the action of perorally administered rChIFN-[alpha] in effect creates super-sentinel chickens that seroconvert in response to levels of antigen that would otherwise go undetected. Super-sentinel chickens would thus provide a novel means of detecting otherwise inapparent infections of LPAI, thereby buying time for its control or eradication.

We envision the introduction into a large flock of a number of small cages containing chickens in which IFN-water replaces plain water. These super-sentinel chickens will serve as sensitive early detectors of LPAI, like the proverbial canary used in mines to detect low levels of toxic gases. Because of the cross-reaction between chicken and turkey IFN-[alpha] (5,13), super-sentinel turkeys could likely be created in a similar manner. Super-sentinel birds could be replaced every month and possibly returned to production.

All strains of chickens tested, including those in the People's Republic of China, have proved to be sensitive to the action of rChIFN-[alpha] (14). Genetically engineered production of rChIFN-[alpha] (15), treatment with it optimized for dose and duration, and its long half-life in water may make it economically feasible to convert many birds in a flock to super-sentinel status. It also may be prudent to set up super-sentinel birds in areas of high risk for avian influenza virus outbreaks, such as live-bird markets. Surveillance of other families of birds might be possible with species-specific IFN. Further studies are required to test these possibilities and the extent to which rChIFN-[alpha] functions as an adjuvant with other strains of avian influenza virus and chickens.

Acknowledgments

We thank David L. Suarez for the quantitative real-time reverse transcriptase--PCR assays.

This research was supported by US Department of Agriculture grant 58-1940-0-007 through the Center of Excellence for Vaccine Research at the University of Connecticut. The study benefited from the use of the Animal Cell Culture Facility of the Biotechnology/Bioservices Center at the University of Connecticut.

References

(1.) Cardona C. Low-pathogenicity avian influenza virus outbreaks in commercial poultry in California. In: The threat of pandemic influenza: are we ready? Washington: National Academies Press; 2005. p. 243-53.

(2.) Chin PS, Hoffman E, Webby R, Webster RG, Guan Y, Peiris M, et al. Molecular evolution of H6 influenza viruses from poultry in southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry. J Virol. 2002;76:507-16.

(3.) Lee C-W, Swayne DE, Linares JA, Senns DA, Suarez DI. H5N2 avian influenza outbreak in Texas in 2004: the first highly pathogenic strain in the United States in 20 years? J Virol. 2005;79:11412-21.

(4.) Kuiken T, Leighton FA, Fouehier RAM, LeDuc JW, Peitis JSM, Schdel A, et al. Pathogen surveillance in animals. Science. 2005;309:1680-1.

(5.) Sekellick MJ, Ferrandino AF, Hopkins DA, Marcus PI. Chicken interferon gene: cloning, expression, and analysis. J Interferon Res. 1994;14:71-9.

(6.) Marcus PI, van der Heide L, Sekellick MJ. Interferon action on avian viruses. I. Oral administration of chicken interferon-[alpha] ameliorates Newcastle disease. J Interferon Cytokine Res. 1999;19:881-5.

(7.) Pei J, Sekellick MJ, Marcus PI, Choi I-S, Collisson EW. Chicken interferon type I inhibits infectious bronchitis virus (IBV) replication and associated respiratory illness. J Interferon Cytokine Res. 2001;21:1071-7.

(8.) Mo CW, Cao TC, Lira BL. The in vivo and in vitro effects of chicken interferon-alpha on infectious bursal disease virus and Newcastle disease infection. Avian Dis. 2001 ;45:389-99.

(9.) Lu H, Castro AE. Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian influenza virus in specific-pathogen-free chickens. Avian Dis. 2004;48: 263-70.

(10.) Sekellick MJ, Carra SA, Bowman A, Marcus PI. Transient resistance of influenza virus to interferon action attributed to random packaging and activity of NS genes. J Interferon Cytokine Res. 2000;20: 963-70.

(11.) Cauthen AN, Swayne DE, Sekellick MJ, Marcus PI, Suarez DL. Amelioration of influenza pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene. J Virol. 2007;81:1838-47.

(12.) Beilharz MW, Fleischmann WR Jr, editors. Special topics issue: oral use of interferons and cytokines. J Interferon Cytokine Res. 1999;19:813-979.

(13.) Suresh M, Karaca K, Foster D, Sharma JM. Molecular and functional characterization of turkey interferon. J Virol. 1995;69:8159-63.

(14.) Xia C, Liu J, Wu ZG, Lin CY, Wang M. The interferon-alpha genes from three chicken lines and its effects on H9N2 influenza viruses. Anim Biotechnol. 2004; 15:77-88.

(15.) Schultz U, Rinderle C, Sekellick MJ, Marcus PI, Staeheli P. Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active. Eur J Biochem. 1995;229:73-6.

Philip I. Marcus, * Theodore Girshick, ([dagger]) Louis van der Heide, * and Margaret J. Sekellick *

* University of Connecticut, Storrs, Connecticut, USA; and ([dagger])Charles River Specific Pathogen Free Avian Supplies, Storrs, Connecticut, USA

Address for correspondence: Philip I. Marcus, Department of Molecular and Cell Biology, University of Connecticut, U-3125, 75 North Eagleville Rd, Storrs, CT 06269, USA; email: [e-mail]

Dr Marcus is professor of molecular and cell biology at the University of Connecticut, Storrs. He has a long-standing interest in the chicken interferon system, including its cloning, expression in the developing embryo, and the sensitivity of influenza viruses to the antiviral action of interferon.

Table 1. Seroconversion in influenza A virus-infected
3-week-old chickens given water or water + interferon *

                                       No. chickens
                                          Water +
Infection                               interferon
type ([dagger])         Water        ([double dagger])   Total

Overtly infected   4/8 ([section])          8/8           16
Sentinel                2/32               31/31          63
Combined                6/40               39/39          79

* Represents 4 independent trials.

([dagger]) Overtly infected birds were mixed with uninfected
sentinel cage mates, the latter to become infected naturally.

([double dagger] Recombinant chicken interferon-a at 2,000
U/mL (5).

([section]) No. positive birds/total no. receiving treatment,
scored by agar gel precipitin or hemagglutination inhibition
tests.

Table 2. Influenza A virus infectious particle equivalents (IPE)
in tracheal swabs from sentinel chickens given water or water +
interferon and infected naturally *

                                            Water + interferon
Day postinfection      Water (IPE/mL)      ([dagger]) (IPE/mL)

2                   1,112 [+ or -] 1,353     760 [+ or -] 632
                     ([double dagger])
4                   1,234 [+ or -] 764       463 [+ or -] 484
10                  1,325 [+ or -] 398     2,113 [+ or -] 1,834

* Each cage contained 2 overtly infected birds and 8 cage mates as
sentinels. Only sentinel birds are reported. Chickens were 3 weeks
old at the start.

([dagger]) Recombinant chicken interferon-a in water at 2,000 U/mL (5).

([double dagger]) Mean [+ or -] SD, n = 8. Quantitative real-time
reverse transcriptase-PCR analysis with influenza A virus standard:
IPE/mL.