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In avian diseases coccidiosis is caused by protozoan parasites of Emeria genus is very important and economically devastating diseases throughout the world. There are seven species of Emeria , E. tenella, E. maxima, E. acervulina, E. mitis, E. necatrix, E. mevati E. burneti. Among all species of Emeria, E. tenella, E. maxima, and E. acevulina are most encountered in field and each specie infects a specific part of intestinal tract. The economic losses are estimated at $ 3 billion annually worldwide (Williams, 1999; Shirley et al., 2004). These estimates include the costs of prophylactic in feed medication, alternative treatments if medication fails, losses due to mortality, morbidity, impaired growth rate, reduction to egg production and poor feed conversion that survive outbreaks (Kitandu and Juranova, 2006). In Pakistan the combination of heat and humidity often encountered the survival of the oocyst to cause disease. The prevalence of coccidiosis in Pakistan is very high as 32 % (Ayaz et al., 2003) although the exact figure is not known due the lack of statistics but these may be probable in millions of rupees. The disease prevalence is wide spread in the poultry industry due to increasing incidence of drug resistance in the parasite despite the use of integrated management system. In Pakistan the prevalence is very high (Ayaz et al., 2003) and cause severe economic losses to the poultry industry.
Various controlled methods against coccidiosis have been employed such as management of poultry house, application of anticoccidials and vaccination (Allen and Fetterer, 2002). Although continuous medication has proven to be highly effective in controlling coccidiosis in modern intensive poultry production system (Chapman, 1999; Allen and Fetterer, 2002), there are several disadvantages related to this strategy. These include cost, variable withdrawal periods, drug residues in meat which is for human consumption, lower dosage of drugs, and short life cycle of parasites (Riddel, 1984) and development of drug resistant strains (Dalloul and Lillehoj, 2005) necessitating a continuous search for more effective anticoccidials. The future of the chemotherapy is questionable due to wide spread emergence of the of drug resistance in the parasites as well as to high costs attributed to discovery, characterization, development and registration of new drugs (Long and Jeffers, 1986). Chemotherapeutics used are generally classified as synthetics and ionophorous antibiotics (compound produces through microbial fermentation) (Chapman, 1990). Ionophore anticoccidials used for treatment of coccidiosis are semduramicin, salinomycin, monensin, narasin, maduramicin, lasalocid and chemical and chemical + ionophore anticoccidials are nicarbazin, amprolium, robenidine, zoalene, clopidol, diclazuril, nicarbazin + narasin. Most commonly chemotherapeutics strategies include the complete suppression of the disease, treatment of outbreaks and partial suppression of the disease for immunity development in layer flocks. Usually anticoccidial resistance arises due to continued use of drugs, use of suboptimal doses of drug, as well as short life cycle of the parasites (Riddell, 1984). Importantly European Parliament is trying to phase out the use of anti-coccidial substances by year 2012 (Martin et al., 2007).
In the past different efforts had been made to immunize the chickens against coccidiosis by using live, attenuated, killed and irradiated vaccines. Commercial vaccines namely Immunocox, Paracox, Coccivac, Livacox, Viracox and COX ATM have been used to control coccidiosis in several countries. There has been a general limitation to use these vaccines in broilers and heavy roaster birds because of reduced weight gain, low feed conversion ratio, risk of introducing unwanted Emeria species into the environment as there is regional variation in the antigenicity of coccidial strains (Martin et al., 1997). Hence, effective and more economically viable vaccines for controlling the Eimeria species are still required. Further certain strains in the live vaccines could affect their efficacy. A better understanding of strain variation is needed for a vaccine development that provides protection against local field strains of Emeria. Recently a new subunit vaccine CoxAbic against coccidiosis has been commercialized and is being used in some parts of the world. This vaccine is based on native gametocytes isolated from E. maxima. It is a novel vaccine that unlike other available vaccine is based on the concept of transmission blocking immunity (Wallach et al., 1995: Wallach, 1997, 2002). It is observed that chicken vaccinated with CoxAbic protect at least as well as the coccidiostat fed broiler controls (Michael, 2002). However the production of CoxAbic is expensive, laborious, and time consuming because it relies on the affinity purification of native gametocyte antigens from their intracellular locations with in the intestine of chicken (Belli et al., 2004). Live vaccines consisting of virulent and attenuated Eimeria parasites have drawbacks including the potential reversion to virulence and high production expenses (Vermeulen, 1998; Du and Wang, 2005).
Several reports provide evidence that sporulated oocysts of Eimeria species gave protection against heavy doses of challenges; further the vaccinated birds revealed a significant cellular and humoral responses (Akhtar et al., 1998; 1999; 2000; 2001; 2001a; 2001b; 2003; Ayaz, 1999; Khan, 1999; Ayaz et al., 2002; 2002a).
Recently egg propagated E. tenella (local isolate) gametocyte vaccine gave protection against mixed species (mainly E. tenella, E. maxima, and E. acervulina) of coccidian in chickens (Ayaz,2003: Hafeez et al., 2006). In this study egg propagated gametocytes of Eimeria tenella (local isolates) were used to prepare the adjuvanted (Amphigen) and nonadjuvanted vaccine(s) and evaluated on the basis of cellular, humoral, and challenge responses. Maximum percent protection (survivors after challenge) against mixed species of genus Eimeria was observed in adjuvanted vaccine (orally) group (A) (71.42%) followed by nonadjuvanted vaccine (orally) group (C) (63.63%), adjuvanted vaccine (subcutaneously, s/c) group (B) (59.09%), and nonadjuvanted vaccine s/c group (D) (54.54). Maximum percent reduction in OPG was also recorded in group A (86) followed by group C (84), group B (83), and group D (82). From these results, it was concluded that egg-propagated gametocytes (E. tenella) that gave protection upon challenge may be due to the control of E. tenella.
This egg propagated vaccine is used in breeding hens (Hafeez et al., 2007). Breeding hens were immunized with adjuvanted gametocyte vaccine orally; upon challenge with sporulated oocysts of E. tenella (local isolates) to 7-day-old chicks from immunized hens showed more than 70% reduction in oocyst production compared with chicks from control hens. Furthermore no mortality was recorded in immunized chickens.. It can help to reduce replication and shedding of the Emeria, and hence reduce overall flock exposure. Antibodies, including IgG, IgA and IgM are secreted during egg formation. Chicks born to immunized hens were reduce oocysts outputs that increased with age as the immunized the maternal antibodies decreased. Antibodies to these surface antigens may limit disease by inhibiting the growth development and fertilization of gametes.
Anwar et al., (2008a) tried the egg adapted gametocyte vaccine in the field and its comparative efficacy with commercial anticoccidial vaccine, LivaCox, is measure and it is used in breeder and broiler flocks in Pakistan. Humoral immune response in vaccinated and control chickens was monitored by enzyme-linked immunosorbent assay. Significantly higher antibody titres in local gametocyte-vaccinated group as compared to LivaCox-vaccinated chickens were recorded. Splenic cell migration inhibition assay was used to detect the cell-mediated immune (CMI) response, and results were expressed in terms of per cent migration index. Lower per cent migration index in LivaCox-vaccinated chickens indicated the higher CMI response, as compared to local gametocyte-vaccinated chickens. Results of the challenge studies in laboratory experiments revealed significantly higher oocyst count in LivaCox-vaccinated group as compared to local gametocyte-vaccinated chickens. Maximum protection (75%) against mixed species of genus Eimeria was recorded in chickens vaccinated with gametocyte vaccines as compared to LivaCox-vaccinated group. The mean body weight gains in chickens vaccinated with local gametocyte vaccine were significantly better than in chickens vaccinated with LivaCox vaccine, both in laboratory and field experiments.
Another study (Anwar et al., 2008) was conducted to see the effects of local gametocyte and livacox vaccines on live body weight gain and lymphoid organs in chickens. Organ to body weight ratio in chickens is considered to be an important parameter to study the immune status due to any infection or vaccination. The mean body weight gains in chickens with local gametocytes vaccine were significantly better than in chickens with Livacox vaccine. Higher organ to body weight ratio was recorded in vaccinated chickens compared to control. Organ to body weight ratio of the lymphoid organs had higher values in chickens vaccinated with Livacox compared to local gametocyte vaccinated group. It was concluded that local gametocyte vaccine significantly increased the body weights of chickens compared to Livacox and control groups.
For the control of coccidiosis different methods are adopted, like chemotherapy, live attenuated and killed vaccines, but we are lacking in the production Eimeria DNA vaccines. Different studies have been conducted in the production of DNA vaccine and they are very good. DNA vaccines have been experimentally tried and successfully developed against coccidiosis. For developing DNA vaccines, generally the immunogenic protein of a pathogen which is protective to the host is incorporated into a suitably designed plasmid which has been derived from Eimeria agents. This gene encoded plasmid DNA, when administered to host, is capable of getting transcribed and translated into a peptide within the host cells to generate protective responses on encountering with the host immune cells. The application of genetic engineering along with other new technologies has played crucial roles in introducing novel ideas in vaccinology. New generation vaccines like subunit vaccines and recombinant DNA vaccines are rapidly gaining acceptance as new generation vaccines, and considered as alternatives to the conventional vaccines. The last decade have seen the development of recombinant plasmid based DNA or nucleic acid vaccines, using immunogenic genes of avian pathogens e.g. coccidiosis. The advantages of DNA vaccine production are;
Non-requirement of cold chain when compared to commonly used live vaccines in poultry.
Provides long-lived cellular and humoral immune response.
Bacterial plasmids used as vector have inherent immunogenic properties.
Can be easily transported in lyophilized form.
Shows enormous stability as a vaccine.
They comprise of circular bacterial plasmids in which desired antigenic gene of avian pathogens are encoded
Even though adjuvants like cytokines such as interleukin (IL)-2, 12, 15, 18, IFN-γ are used in combination with DNA vaccine as new generation adjuvants. Also, the utility of co-stimulatory molecules, which stimulates the generated specific immune cells.
Activation of immunity more inclined towards cellular immune responses when compared to humoral responses, especially useful to control intracellular pathogens.
Contrary to the direct generation of humoral or cellular immunity as seen in case of killed or live vaccines, the DNA vaccines enter into muscle cells produce the immunogenic pathogen proteins for further action of the immune system.
The results of some DNA vaccine studies are discussed below;
DNA immunization induces both antigen-specific antibody and cytotoxic T-cell responses and elicits protection against bacteria, virus and parasites (Manickan et al., 1997, Tang et al., 1992). Chickens immunized with 5, 10, 50 and 100 ug of pMP13 (recombinant plasmid) showed a significantly reduced oocyst production compared to pBK-CMV-immunized chickens. Two injections with 10 or 100 ug DNA were more effective in reducing oocyst production compared to single injection (Song et al., 2001). The level of anti-3-1E antibody responses depended on the dose of plasmid DNA as well as on the frequency of immunization. At 2 weeks post-immunization with pMP13, 100 ug (pMP13) del plasmid DNA induced higher levels of antibodies than 10 ug DNA. Furthermore, two immunizations consistently induced higher levels of antibodies than one immunization (Song et al., 2001). DNA immunization induced significant changes in T lymphocytes in the spleen and duodenum. At 4 days post DNA immunization, the percentage of duodenum IEL CD8a cells decreased in pMP13-immunized chickens compared to pBK-CMV-immunized chickens whereas duodenum CD8b IEL increased in the pMP13-immunized chickens (Song et al., 2001).
Min et al,. (2001) examined the effects of injecting a plasmid encoding the 3-1E gene in combination with a plasmid encoding IL-1b, IL-2, IL-8, IL-15, IFN-a, IFN-c, TGFb4, or lymphotactin and delivered twice subcutaneously to chickens, followed by challenge 1 wk later. Body weight loss was significantly reduced in chickens given the DNA vaccine with the IFN-a or the lymphotactin-encoding plasmid, whereas parasite replication was reduced in chickens injected with the IL-8, lymphotactin, IFN-c, IL- 15, TGF-b4, or IL-1b-encoding plasmids, compared with chickens vaccinated with the 3-1E DNA vaccine alone.
Synthetic peptide vaccines may allow the species-specific immunity barrier to be overcome in the design of broad-spectrum anti-coccidial vaccines (Talebi et al., 2005). This study demonstrated that peptides are capable of eliciting high antibody responses and relatively good proliferation of lymphocytes in chickens to Eimeria species, resulting in partial cross-species protection against challenge with E. acervulina and E. tenella.
A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-γ. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina (Lillehoj et al., 2005). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-γ genes induced higher serum antibody responses with immunization. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-γ significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis. Song et al. (2009) and Lillehoj et al. (2005a) documented that vaccination of chickens with cloned Eimeria gene plus IL-2, IL-15 and IFN-γ stimulated more protective intestinal immunity against coccidiosis.
TA4 gene of E. tenella and chIL-2 gene were cloned into expression vector pcDNA3.1 and pcDNA4.0c in different forms, producing vaccines pcDNA3.1-TA4-IL-2, pcDNA3.1-TA4 and pcDNA4.0c-IL-2. DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. DNA vaccines could obviously alleviate caecal lesions, body weight loss and increase oocyst decrease ratio. The results suggested that TA4 was an effective candidate antigen for vaccine and co-expression of cytokine with antigen was an alternative method to enhance DNA vaccine immunity (Xu, et al., 2008).
Shah et al., (2010) describes cross protection experiments with chimeric DNA vaccine pVAX1-cSZ2-IL-2 to determine its efficacy against four important Eimeria species. The results indicated that the recombinant plasmid can induce host immune responses by alleviating intestinal lesions, body weight loss and oocyst ratio and imparting good protection against E. tenella and E. acervulina, medium protection against E. necatrix. A recombinant DNA vaccine encoding Eimeria acervulina cSZ-2 induces immunity against experimental E. tenella infection (Shah et al., 2010a) make it obvious that cSZ-2 DNA immunisation can induce host immune responses by decreasing intestinal lesions, body weight loss and oocyst ratio, imparting partial protection.