Elsevier

Vaccine

Volume 29, Issue 49, 15 November 2011, Pages 9201-9208
Vaccine

Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis

https://doi.org/10.1016/j.vaccine.2011.09.117Get rights and content

Abstract

Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge.

Highlights

Eimeria tenella Microneme 2 (EtMIC2) gene was clones from sporozoite RNA. ► The gene was transiently expressed in tobacco and characterized immunologically. ► Efficacy of recombinant EtMIC2 in bird challenege experiment was evaluated. ► Plant expressed antigen confer partial protection to immunized birds.

Introduction

Avian coccidiosis is an economically important disease of poultry caused by protozoa of the genus Eimeria. Eimeria tenella is one of the seven different Eimeria species that infect chicken. The infection of Eimeria species causes damage to the intestinal epithelium with varying severity accompanied by reduction in body weight, reduced feed conversion efficiency and shedding of parasite oocysts in feces. Use of ‘coccidiostat’ and ‘coccidiocidal’ chemicals in poultry feed is the popular method of managing coccidiosis. However, the emergence of drug resistant Eimeria strains is of serious concern [1]. In order to contain the indiscriminate use of coccidiostat, European parliament has set a target of 2012 to phase out the use of coccidiostat in poultry feed [2]. Live vaccines containing either virulent or attenuated strains of Eimeria or affinity purified oocysts antigens are available as alternatives to chemo-prophylaxis. However, their use is restricted to Breeder and Layer stocks in the poultry industry as the vaccines are produced in very limited quantity [3]. Eimeria infection involves multiple stages of parasite invasion [4]. One of the approaches in developing a prophylactic vaccine, explored by many investigators, is to block the parasite invasion into gut epithelium. Microneme organelles are located at the apical tip of invading stage of all apicomplexan parasites and they harbor several proteins that are critical for motility of the parasite, identification and binding of the host cell-–surface proteins and invasion of host cells [5]. Thus, induction of neutralizing antibodies to one or several of these ‘invasion proteins’ presents a rational approach in developing a prophylactic vaccine. Earlier reports suggest that the recombinant microneme antigens might protect chickens against coccidiosis when used as vaccine [4], [6].

Plants have been used to express a wide variety of proteins for therapeutic and diagnostic use. The plant expression platform has been particularly attractive because of the ease of transformation, low investment, high and controlled level of expression, easy scale up with no process optimization downtime, etc. Plants have the ability of performing post-translational modifications and complete absence of parasite/pathogens that may harm animals or humans makes plant particularly attractive expression host for therapeutic proteins [7], [8], [9], [10]. We have explored the possibility of using plant expression system for production of prophylactic vaccine against poultry coccidiosis.

Here, we report transient expression of one of the microneme proteins, EtMIC2 from E. tenella as His6-tagged fusion protein, in tobacco using Agro-infiltration. Chicken were immunized using the plant expressed EtMIC2 protein and humoral and cell-mediated immune responses in the immunized birds were measured. Protective efficacy of the plant-expressed EtMIC2 antigen was also evaluated. The findings of our studies hold promise for developing a low cost sub-unit coccidiosis vaccine for the poultry industry.

Section snippets

Chicken

Day-old, coccidiosis free, male White Leghorn layer chickens (commercial breed—BV 300) were obtained from Sri Venkateswara Hatcheries (Hyderabad, India) and reared in clean brooder cages. The birds were provided with coccidiostat-free feed and water ad libidum. Birds were shifted to animal containment facility prior to challenge with E. tenella sporulated oocysts.

Coccidial oocysts

Wild type E. tenella oocysts were isolated from an Eimeria-infected farm in India. Oocysts were propagated in 3 weeks old birds by

Construction of Agrobacterium clones containing EtMIC2 expression plasmid

The 1.2 kb full length cDNA of EtMIC2 coding sequence was amplified using RT-PCR from the excysted sporozoite RNA and cloned into plant expression vector pTRA vector. Recombinant clones of EtMIC2 produced a DNA fragment of 1.2 kb upon digestion of pTRAEtM2 with EcoRI and NotI restriction enzymes (Supplementary Fig. 1). Upon sequence comparison with the available EtMIC2 sequences of Houghton strain (GenBank accession number: Z71755) and Beijing strain (GenBank accession number: AF111839) we

Discussion

In order to contain the loss due to coccidiosis, widespread reliance on the in-feed anti-coccidial drugs in poultry industry have created challenges such as the emergence of drug resistant parasites and presence of drug residues in meat and egg, etc. [1], [20]. A number of governments have realized the impact of excessive use of the anti-coccidial drugs by the industry and have enacted legislations limiting/prohibiting the use of the drugs in poultry feed [2], [21]. Thus, effective alternative

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