Michael Wallach

Chasing the golden egg: vaccination against poultry coccidiosis

Eimeria species, of the Phylum Apicomplexa, cause the disease coccidiosis in poultry, resulting in severe economic losses every year. Transmission of the disease is via the faecal‐oral route, and is facilitated by intensive rearing conditions in the poultry industry. Additionally, Eimeria has developed drug resistance against most anticoccidials used today, which, along with the public demand for chemical free meat, has lead to the requirement for an effective vaccine strategy. This review focuses on the history and current status of anticoccidial vaccines, and our work in developing the transmission‐blocking vaccine, CoxAbic® (Netanya, Israel). The vaccine is composed of affinity‐purified antigens from the wall‐forming bodies of macrogametocytes of Eimeria maxima, which are proteolytically processed and cross‐linked via tyrosine residues to form the environmentally resistant oocyst wall. The vaccine is delivered via maternal immunization, where vaccination of laying hens leads to protection of broiler offspring. It has been extensively tested for efficacy and safety in field trials conducted in five countries and involving over 60 million offspring chickens from immunized hens and is currently the only subunit vaccine against any protozoan parasite to reach the marketplace.

Eimeria maxima gametocyte antigens: potential use in a subunit maternal vaccine against coccidiosis in chickens

Affinity-purified gametocyte antigens (APGA) from Eimeria maxima, emulsified in Freunds adjuvant, were injected intramuscularly into breeding hens on two or three occasions. As a result, progeny of the immunized hens were partially immune to infection with E. maxima, Eimeria tenella and Eimeria acervulina (with a reduction in total oocyst output of 45-63% as compared with progeny of untreated hens). Thus, APGA in Freunds adjuvant appears to have great potential as part of a maternally applied vaccine against coccidiosis. The ability of APGA to induce partial cross-species protection is most probably due to the existence of conserved epitopes in the different species as indicated by comparative Western blots of E. maxima and E. tenella. Surprisingly, Freunds adjuvant by itself also induced significant levels of maternal immunity to coccidiosis (with a 12-35% reduction in oocyst output in the progeny). In contrast to the purified antigens or Freunds alone, crude extracts from gametocytes as well as other developmental stages, induced little if any significant maternal immunity despite provoking the production of large amounts of parasite-specific IgG, including antibodies to APGA. This result indicates that a successful maternal vaccine against coccidiosis requires, in addition to good recognition of protective antigens, the exclusion of irrelevant antigens from the vaccine preparation.

Maternal transfer of antibodies induced by infection with Eimeria maxima partially protects chickens against challenge with Eimeria tenella

Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20,000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6.8 +/- 1.2 million (mean +/- S.E., n = 10) or 5.8 +/- 1.2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0.9 +/- 0.4 million (n = 5) E. maxima oocysts or 2.2 +/- 0.4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, eggs were collected from days 28 to 37 and again from days 114 to 123 after infection of the hens with E. maxima and hatchling oocyst excretion rates were 82% and 62%, respectively, reduced for E. maxima and 43% and 41%, respectively, reduced for E. tenella in the progeny of hens infected with E. maxima compared to the progeny of uninfected hens. ELISA and Western blot analyses of maternally-derived IgG revealed a high degree of cross-reactivity to antigens of E. maxima and E. tenella.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of Tyrosine-Rich Precursor Glycoproteins and Dityrosine- and 3,4-Dihydroxyphenylalanine-Mediated Protein Cross-Linking in Development of the Oocyst Wall in the Coccidian Parasite Eimeria maxima

ABSTRACT The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.

Eimeria maxima: identification of gametocyte protein antigens.

The antigenicity of Eimeria maxima gametocyte proteins during the course of an infection and when injected into mice and rabbits was demonstrated using the Western blotting technique. Serum taken from chickens at various times postinfection reacted to a few gametocyte proteins, with the strongest reactivity seen with serum taken 14-days postinfection. Two major antigens having molecular weights of 56,000 and 82,000 were consistently detected by these sera. Using immune rabbit or mouse sera to whole gametocyte detergent extracts, the 56,000 and 82,000 molecular weight proteins were again the immunodominant antigens, despite their representing only a small proportion of the extract which was used to immunize the animals. These results, together with those obtained by Rose (1971) using recovered chicken serum to passively immunize chickens, indicate that these two gametocyte antigens may play a role in protective immunity to E. maxima.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

The development of the macrogamete and oocyst wall in Eimeria maxima: immuno-light and electron microscopy.

We have identified, and followed the development of three macrogamete organelles involved in the formation of the oocyst wall of Eimeria maxima. The first were small lucent vacuoles that cross-reacted with antibodies to the apple domains of the Toxoplasma gondii microneme protein 4. They appeared early in development and were secreted during macrogamete maturation to form an outer veil and were termed veil forming bodies. The second were the wall forming bodies type 1, large, electron dense vacuoles that stained positively only with antibodies raised to an enriched preparation of the native forms of 56 (gam56), 82 (gam82) and 230 kDa (gam230) gametocyte antigens (termed anti-APGA). The third were the wall forming bodies type 2, which appeared before the wall forming bodies type 1 but remain enclosed within the rough endoplasmic reticulum and stained positively with antibodies raised to recombinant versions of gam56 (anti-gam56), gam82 (anti-gam82) and gam230 (anti-gam230) plus anti-APGA. At the initiation of oocyst wall formation, the anti-T. gondii microneme protein 4 positive outer veil detached from the surface. The outer layer of the oocyst wall was formed by the release of the contents of wall forming bodies type 1 at the surface to form an electron dense, anti-APGA positive layer. The wall forming bodies type 2 appeared, subsequently, to give rise to the electron lucent inner layer. Thus, oocyst wall formation in E. maxima represents a sequential release of the contents of the veil forming bodies, wall forming bodies types 1 and 2 and this may be controlled at the level of the rough endoplasmic reticulum/Golgi body.

Role of antibody in immunity and control of chicken coccidiosis

Research has been carried out worldwide to try to elucidate the mechanism of protective immunity against coccidiosis. It was concluded from early studies that cellular immunity is the key to protection against Eimeria, whereas humoral immunity plays a very minor role in resistance against infection. By contrast, other studies have pointed towards the ability of antibody to block parasite invasion, development and transmission and to provide passive and maternal immunity against challenge infection. Herein, recent results demonstrate the ability of antibodies (raised by live immunization or against purified stage-specific Eimeria antigens) to inhibit parasite development in vitro and in vivo and readdress the question of the role of antibody in protection against coccidiosis.

The importance of transmission-blocking immunity in the control of infections by apicomplexan parasites

Transmission-blocking immunity may have great potential for use in the control of diseases caused by apicomplexan parasites. In this review I will describe our work on the application of transmission-blocking immunity to the control of the Eimeria parasite and compare our results to those working on transmission-blocking immunity against Cryptosporidium and Plasmodium. Eimeria causes the disease known as coccidiosis in domestic animals. Coccidiosis is particularly problematic in the chicken industry, mainly due to the crowded rearing conditions under which chicks are raised. In our work we identified, isolated and characterized 3 major gametocyte antigens (230 kDa, 82 kDa and 56/54 kDa) of Eimeria maxima. We used these native glycoproteins to immunize laying hens that, via the egg yolk, provide large amounts of transmission-blocking maternal antibodies to offspring chicks. We demonstrated that hatchlings from immunized hens shed 60-80% fewer oocysts (i.e. the infective stage of the life-cycle of Eimeria) than those from control hens. Such a reduction in oocyst output acts to significantly reduce parasite numbers in the litter of chicks raised in floor pens. This reduction in oocyst output is comparable to that seen using the most effective coccidiostat drugs and is probably sufficient to control coccidiosis under field conditions. Based on our results together with those of other groups working on transmission-blocking immunity against Cryptosporidium and Plasmodium, it appears that this immunological approach holds great promise for the control of apicomplexan parasites that cause diseases in both animals and man.

Field Application of a Subunit Vaccine against an Enteric Protozoan Disease

Background Coccidiosis is a major global veterinary health problem in intensively reared chickens. It is caused by apicomplexan parasites of the genus Eimeria. Principal Findings A subunit vaccine composed of purified antigens from the gametocytes of Eimeria maxima was used to stimulate the production and transfer of maternal antibodies between breeding hens and their hatchlings. The vaccine was injected into hens twice before they began laying eggs. Immunization had no adverse affects on egg laying or health of the hens and resulted in high antibody levels throughout the life of the hens. Progeny of immunized hens excreted significantly less oocysts of various species of Eimeria in their faeces than chicks from unvaccinated hens. Furthermore, the offspring of vaccinated hens developed stronger natural immunity to Eimeria, so that they were resistant to challenge infection even at 8 weeks of age, well after all maternal antibodies had left their circulation. Field trials were conducted in South Africa, Brazil and Thailand, involving at least 1 million progeny of vaccinated hens and at least 1 million positive control birds (raised on feed containing anticoccidial drugs or immunized with a live vaccine) in each country. Additionally, trials were carried out in Israel involving 60 million progeny of vaccinated hens and 112 million positive control birds. There were no significant differences in growth rate, feed conversion ratios or mortality in the offspring of vaccinated hens compared with the positive control chickens in any of these countries regardless of different management practices, different breeds of chickens or climate. Conclusions These results demonstrate that a vaccine composed of antigens purified from the gametocytes of Eimeria can be used safely and effectively to prevent the deleterious effects of coccidiosis. It is the first subunit vaccine against any protozoan parasite to be successfully applied on a commercial scale.