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Rural Industries Research & Development Corporation
New Therapeutics for Poultry
Therapeutic applications of chicken interferon gamma (ChIFN-?) in poultryby John W. Lowenthal
November 2001
RIRDC Publication No 01/149 RIRDC Project No CSA-7J
Newly hatched chicks are highly susceptible to infection by opportunistic pathogens during the first two weeks of life. Several factors contribute to this problem; broiler chickens are reared under crowded conditions, their immune system has not yet fully developed and the levels of maternal antibody are rapidly declining. A major problem faced by all livestock industries is reduced productivity due to disease and considerable resources must be applied in order to maintain the health status of animals.
The two main mechanisms by which disease is controlled involves the use of vaccines and antimicrobials. Vaccines aim to offer long term immunity and protection against a particular pathogen following a small number of immunisations. In contrast, broad spectrum protection provided by antimicrobials (as in-feed antibiotics and chemicals) requires their continual usage even in the absence of apparent disease. Antibiotics, in addition to being an antimicrobial, also have growth promoting activity which makes them even more attractive. Unfortunately, the extensive use of antibiotics and chemicals over a long period of time has resulted in the emergence of pathogens that have become resistant to such treatments. The World Health Organisation has now recommended restrictions in the type of antimicrobials used in food production animals and has recommended the development and use of alternative, environmentally-friendly methods to control disease.
Some countries have already implemented such restrictions and based on their experiences, it is anticipated that without appropriate substitutes for prophylactic antibiotics, particular microorganisms may emerge as significant health problems. Faced with these restrictions and potential problems, the Australian poultry industry is supporting the development of alternative measures that will maintain productivity as well as ensuring the highest possible levels of animal health and welfare.
Another major problem faced by the poultry industry is related to vaccines which are designed to give high levels of protection to specific diseases. Vaccination strategies are the primary mechanism for the control of most parasitic, viral and bacterial pathogens. There are, however, concerns over the ability of current live vaccines to protect against emerging hyper-virulent strains of pathogens. For certain diseases, there is a need for alternative vaccines, however, killed and recombinant subunit vaccines do not usually offer an adequate level of long term protection and often require the use of adjuvants to enhance their activity. Oil-based adjuvants, however, induce adverse site reactions resulting in decreased meat quality and animal discomfort and are therefore not used. At this time there is a lack of suitable, cost effective adjuvants for use in both the broiler and egg industries.
Cytokines are proteins that control immune responses following infection or vaccination and represent excellent, naturally occurring therapeutics. The efficacy of cytokine therapy has been demonstrated in several human and animal studies. Cytokine therapy has been successfully used in humans for the treatment of immunodeficiencies, in particular, patients suffering from an impaired immune system as a result of cancer treatment have had their immune responses restored following administration of particular types of cytokines called colony stimulating factors. This enabled them to combat pathogenic organisms that would have otherwise overwhelmed them. This is a situation analogous to the immunodeficient nature of newly hatched chickens. The utilization of cytokines is becoming more feasible with the recent cloning of a number of cytokine genes and the establishment of commercially feasible methods of delivery. There are many different types of cytokines that perform different functions. Also, cytokines from one species cannot function in another species, therefore a number of chicken cytokines must be identified, characterized and assessed in order for the most beneficial ones to be identified.
In order to establish a proof-of-principal, we have assessed one of the most characterised chicken cytokines, interferon gamma (ChIFN-?). ChIFN-? is a member of a family of cytokines that share the capacity to modulate the immune response and inhibit viral replication. We have previously shown that treatment with ChIFN-? resulted in enhanced growth rates in healthy SPF chickens as well as in chickens infected with Eimeria acervulina. Furthermore, when co-administered with antigen, ChIFN-? produced a prolonged secondary antibody response in SPF birds that persisted at higher levels and for longer periods compared to antigen injected alone.
The overall objective of this project was to assess the ability of ChIFN-? to enhance disease resistance and increase vaccine efficacy in commercial broilers and layers. The first objective was to produce and optimize a potential commercial product (recombinant ChIFN-? protein). The second objective was to perform pen trials under commercial conditions to assess the ability of ChIFN-? to increase broiler growth performance, enhance vaccine efficacy and improve disease resistance.
In this project we have developed and compared several expression systems for the production of recombinant ChIFN-? protein. Naturally-occurring ChIFN-? can only be produced in very small amounts so more efficient methods of production are needed. ChIFN-? was produced and purified from several established protein expression systems including plants, insect cells, yeast and bacteria.
The E coli system was judged to be preferable as a potential commercial production system and was used in the animal trials.
We also developed several monoclonal antibodies and poly sera specific for ChIFN-? that were used to develop a sensitive and specific ELISA for the detection and quantitation of ChIFN-?. The ELISA was shown to detect only biologically active ChIFN-? and it’s sensitivity was greater than that of the standard bioassay. This ELISA proved to be an invaluable tool in the assessment of function and stability of ChIFN-? in vivo. We showed that intravenous injection of ChIFN-? protein into birds resulted in a rapid rise in circulating levels of protein followed by a fast decline. Intraperitoneal injection resulted in a slower rise in the level of serum ChIFN-? followed by a slow decline. Both methods of delivery resulted in a biological response to ChIFN-? as measured by increased expression of Class II antigen (a marker for enhanced immune activation by interferon) within 24-48 hr of treatment. For practical purposes, intraperitoneal injection was chosen as the preferred route of delivery for ChIFN-? protein.
Several trials were performed in order to assess the ability of ChIFN-? to enhance the growth performance of broilers reared under commercial conditions. Broilers were injected at day of hatch with ChIFN-? protein and monitored for weight gain over a 8 week period. Treatment resulted in a 2.7% increase in mean body weight at day 56. Two similar trials also showed enhanced productivity following ChIFN-? treatment.
We found that chemical modification of the ChIFN-? protein by polyethylene glycol (PEG) enhanced its in vivo stability compared to unmodified ChIFN-?. We then tested whether such modification was able to further enhance the growth promoting activity of ChIFN-?. Broilers were injected with either PEG-modified or unmodified ChIFN-?, and monitored for weight gain over a 6 week period. Both forms of ChIFN-? were shown to be equally effective, producing a 1.3% increase in mean body weight.
CSIRO had previously developed and patented an effective way of delivering proteins to chickens via the use of fowl adenovirus (FAV) vectors. In a separate collaboration with Dr M Johnson, we have performed parallel experiments to assess the growth promoting activity of ChIFN-? when delivered by FAV. The consistent finding was that broiler chickens treated with FAV::ChIFN-? displayed enhanced weight gain (ranging from 1-7% relative to control birds) over periods of up to 8 weeks. Taken together, these results clearly indicate the potential use of ChIFN-? as an effective, naturally occurring growth promoter. The underlying mechanisms are unknown, but may be due to the ChIFN-? mediated enhancement of the immune system leading to decreased pathogen loads, resulting in healthier and more productive birds.
Several trials were performed in order to assess the effect of ChIFN-? treatment to enhance the growth performance of broilers following a coccidiosis infection. Worldwide, coccidiosis represents the largest single threat to loss of productivity. Results from two representative trials are described in detail in this report. Infection of chickens with Eimeria, the causative agent for coccidiosis, results in a reduction in weight gain for several days. We assessed the ability of ChIFN-? treatment to enhance protection against coccidial infection by measuring its ability to reduce the rate of weight loss associated with infection. Chickens were treated orally with FAV::ChIFN-? at five days of age. A second group was treated with another cytokine, chicken myelomonocytic growth factor (cMGF) which was used as a negative control. All birds were then challenged with a dose of E acervulina that induces weight reduction. Birds treated with cMGF and non-treated birds had a significantly reduced rate of weight gain between days 7 and 11 post challenge, relative to ChIFN-? treated birds. This is an indication that ChIFN-? was able to reduce the severity of the infection. In a similar trial, treatment of infected birds with ChIFN-? resulted in enhanced growth performance relative to non treated infected birds (7.1% and 2.9% increase in body weight at day 35 and 42, respectively).
Eimeriavax3 (Eimeria Pty. Ltd.) is a newly developed live vaccine that confers protection against challenge with E. brunetti in the absence of in-feed coccidial medication. The relatively high cost of production for this vaccine makes it not cost-effective for use in the broiler industry. If the effective dose could be significantly reduced by use of an adjuvant, then this vaccine could be used for broilers. Preliminary trials were conducted to assess the compatibility of ChIFN-? co-treatment and vaccination. Birds were vaccinated with Eimeriavax3 either with or without ChIFN-? co-treatment and then challenged 3 weeks later with E. brunetti at a dose that reduced growth rate in unprotected birds. Unvaccinated control birds displayed a decline in growth rate one week after Eimeria challenge. The growth rate of vaccinated birds on the other hand was not affected, as was the case for the vaccinated birds that were co-treated with ChIFN-?, indicating that co-treatment did not reduce the protective effect of the vaccine. These results will allow future experiments to be performed to test whether treatment with ChIFN-? will allow a lower dose of Eimeriavax3 to be used.
As expected with a live vaccination, there was a high output of oocysts beginning 5 days after vaccination and declining to very low levels within 12 days post-vaccination. ChIFN-? did not alter oocyst output. Oocysts were not detected in the faeces of unvaccinated birds prior to challenge, but at 7 days post challenge there was a high output measured in this group. In contrast, the output in both of the vaccinated groups was approximately 1000-fold lower, indicating the effectiveness of the vaccination.
The results from this project have significant implications for the Australian poultry industry. Future restrictions in the use of in-feed antibiotics will have an economic impact on the industry unless alternative therapeutics are developed. Based on overseas experience, pathogens that are currently kept under control by antibiotics, such as Clostridium species, will become more prevalent, leading to severe cases of necrotic enteritis and other gastrointestinal problems. There is a risk that incidence of associated secondary infections will increase significantly, resulting in increase costs due to medication and vaccines and reduced productivity.
Alternative therapeutics such as cytokines and bacteriocins are currently being assessed as safe, naturally occurring alternatives to in-feed antibiotics. Cytokines offer a two pronged attack. They can strengthen and control immune responses resulting in the increase in the general health of animals.
Secondly, they can improve the efficacy of vaccines, resulting in improved long term protection against specific pathogens. In this project we have made some very significant steps forward in the evaluation of chicken cytokines as alternative therapeutics. We have established proof-of-principal by showing that ChIFN-? is a growth promoter and immunoenhancer under commercial conditions. The introduction of cytokines as commercial therapeutics should compensate for the reduction in the use of in-feed growth promotants. As a measure of the successful outcome of this project, it is anticipated that ChIFN-? technology will be implemented in the Australian market in the near future. An Australian poultry health company is currently negotiating with CSIRO for a licence to market ChIFN-? technology to the Australian poultry industry.
To best make use of the outcomes of this project, the following recommendations are made for further work required in this area:
1. Evaluation of additional
cytokines to provide an extended range of therapeutics.
2. Identification of the
nature of protective immune responses to infection as a rational approach
for identifying potential therapeutics.
3. Development of optimal
methods for the safe and efficient delivery of therapeutics under commercial
conditions.
4. Combined use of a variety
of complimentary approaches (cytokines, bacteriocins and vaccines).
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