Super-sentinel chickens and detection of low-pathogenicity influenza virus.Chicken interferon-[alpha] administered perorally in drinking water drinking water
supply of water available to animals for drinking supplied via nipples, in troughs, dams, ponds and larger natural water sources; an insufficient supply leads to dehydration; it can be the source of infection, e.g. leptospirosis, salmonellosis, or of poisoning, e.g. acts on the ompharyngeal mucosal system as an adjuvant adjuvant /ad·ju·vant/ (aj?dbobr-vant) (a-joo´vant)
1. assisting or aiding.
2. a substance that aids another, such as an auxiliary remedy.
3. 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 inapparent
not clearly seen.
infection without clinical signs. infections.
Early detection of low-pathogenicity type A influenza virus (LPAI LPAI Low Pathogenic Avian Influenza ) 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 HPAI Highly Pathogenic Avian Influenza
HPAI Hospital Pharmacists Association, Ireland
HPAI Hewlett Packard Associates International ) (2), and 3) the H5 and H7 LPAI strains can mutate mu·tate
intr. & tr.v. mu·tat·ed, mu·tat·ing, mu·tates
To undergo or cause to undergo mutation.
[Latin m 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 se·rol·o·gy
n. pl. se·rol·o·gies
1. The science that deals with the properties and reactions of serums, especially blood serum.
2. 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.
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 tracheal
pertaining to or emanating from trachea.
see transtracheal aspiration.
tracheal band sign
on contrast radiography of a dilated esophagus, the impression made ventrally by the trachea. samples were sent to a diagnostic laboratory to confirm the diagnosis of LPAI (H7N2) infection at National Veterinary Services Laboratory (NVSL NVSL National Veterinary Services Laboratories (United States Department of Agriculture)
NVSL Northern Virginia Swimming League ) (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 peroral /per·oral/ (per-or´al) performed or administered through the mouth.
Performed or administered through or by way of the mouth. administration of rChIFN-[alpha] under conditions found to ameliorate Newcastle disease (6), infectious bronchitis (7), and infectious bursal disease Infectious Bursal Disease (IBD) is a highly contagious disease of young chickens caused by infectious bursal disease virus (IBDV), characterized by immunosuppression and mortality generally at 3 to 6 weeks of age. (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 (1) (Stateful Packet Firewall) See stateful inspection.
(2) (Sender Policy Framework) An e-mail authentication system that verifies that the message came from an authorized mail server. ) white leghorns (Charles River Specific Pathogen Free specific pathogen free
a term applied to animals reared for experimentation or to commence new herds or flocks of disease-free animals; abbreviated SPF. Animals usually obtained as for axenic animals but are then placed into a nonsterile environment in which they become infected 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 inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula material used in inoculation.
n. pl. 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 ingestion /in·ges·tion/ (-chun) the taking of food, drugs, etc., into the body by mouth.
1. The act of taking food and drink into the body by the mouth.
2. 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 IFN
IFN Interferon, see there ) 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 precipitin /pre·cip·i·tin/ (-sip´it-in) an antibody to soluble antigen that specifically aggregates the macromolecular antigen in vivo or in vitro to give a visible precipitate.
n. (AGP (Accelerated Graphics Port) A high-speed 32-bit port from Intel for attaching a display adapter to a PC. It provides a direct connection between the card and memory, and only one AGP slot is on the motherboard. ) tests to detect antibody against avian influenza virus nucleoprotein nucleoprotein
Macromolecular complex consisting of a protein linked to a nucleic acid, either DNA or RNA. The proteins that combine with DNA are generally of characteristic types called histones and protamines. 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 seropositive /se·ro·pos·i·tive/ (-poz´i-tiv) showing positive results on serological examination; showing a high level of antibody.
adj. 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 hemagglutination /he·mag·glu·ti·na·tion/ (he?mah-gloo-ti-na´shun) agglutination of erythrocytes.
n. 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 seroconversion /se·ro·con·ver·sion/ (-con-ver´zhun) the change of a seronegative test from negative to positive, indicating the development of antibodies in response to immunization or infection. . 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]
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.
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 University of Connecticut is the State of Connecticut's land-grant university. It was founded in 1881 and serves more than 27,000 students on its six campuses, including more than 9,000 graduate students in multiple programs.
UConn's main campus is in Storrs, Connecticut. . The study benefited from the use of the Animal Cell Culture Animal Cell Culture
The process of culture of animal cells in an environment outside the tissue (ex vivo) from which it is obtained is called Animal Cell Culture (ACC). Facility of the Biotechnology/Bioservices Center at the University of Connecticut.
(1.) Cardona C. Low-pathogenicity avian influenza virus outbreaks in commercial poultry in California. In: The threat of pandemic pandemic /pan·dem·ic/ (pan-dem´ik)
1. a widespread epidemic of a disease.
2. widely epidemic.
Epidemic over a wide geographic area.
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JSM Just Shoot Me (sitcom)
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JSM Journal of Software Maintenance
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JSM John Sidney McCain
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Any of a group of soluble proteins that are released by a cell to send messages which are delivered to the same cell (autocrine), an adjacent cell (paracrine), or a distant cell (endocrine). Res. 1999;19:881-5.
(7.) Pei J, Sekellick MJ, Marcus PI, Choi I-S I-S Information System(s)
I-S Instructor-To-Student (Ratio) , Collisson EW. Chicken interferon type I Human type I interferons comprise a vast and growing group of IFN proteins.
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IBV Information Behaviors and Values
IBV Ibm Institute for Business Value ) replication and associated respiratory illness. J Interferon Cytokine Res. 2001;21:1071-7.
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(11.) Cauthen AN, Swayne DE, Sekellick MJ, Marcus PI, Suarez DL. Amelioration a·me·lio·ra·tion
1. The act or an instance of ameliorating.
2. The state of being ameliorated; improvement.
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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: email@example.com
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.