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Amino acid sequence developed
in the laboratory destroys the parasite that causes coccidiosis

Unicamp creates substance
for farm disease control

AGNALDO BRITO

Deleven amino acids in a precise combination resulted in a scientific discovery that could save the third most important activity in Brazilian agribusiness from a loss of approximately US$ 60 million per year. Avian coccidiosis, a disease of economic importance that causes a decrease in the normal growth efficiency of broiler chickens, can be treated without the use of conventional medicines, a strategy that begins to show fatigue. Strains of the Eimeria parasite are already partially or completely resistant to medicines administered by commercial farms. The new technique, being developed at Unicamp, also allows the production of animals that are less subject to iimposed by European and Asian importers, who already veto access to the market for products with residues of medicinal substances in chicken meat. The restriction is one of the important trade barriers to access the European Union and the Asian market.

For Brazil, the discovery is more than promising. Despite the disease, national production of broiler chickens in the country has grown at a dizzying pace. In 2001, chicken production - according to data from the Brazilian Agriculture and Livestock Confederation (CNA) - was 6,73 million tons. It rose to 7,51 million tons in 2002 and could reach 7,89 million tons this year, a volume that should offer the sector a gross production value (VBP) - revenue - of R$ 12,6 billion . The fight against the disease can further increase this result in the coming years.

Researchers from the Center for Molecular Biology and Genetic Engineering (CBMEG) and the Parasitology Department of the Unicamp Biology Institute managed to create in the laboratory a peptide, called PW2 - in short, a sequence of amino acids -, capable of breaking the protective membrane of the protozoan before starting the process of asexual reproduction in the chicken's intestine. The reproduction rate in the intestine is accentuated. It varies depending on the species of the parasite. In total, there are seven species of the Eimeria parasite capable of infecting chicken. The most common on farms in the main producing countries, including Brazil, are Eimeria acervulina, Eimeria maxima and Eimeria tenella.

According to Arnaldo da Silva Júnior, responsible for the doctoral thesis that resulted in the discovery of the amino acid sequence, the initial strategy was to attack the protozoan before it invaded the animal's intestinal cell. When passing through the chicken's digestive system, the oocyst used by the parasite for development in the environment is crushed. From then on, the sporozoite, the first form of the protozoan, travels through the animal's intestine, identifying the attachment region and then penetrating the cell. To do this, the parasite's membrane expels a substance to promote this adhesion, moments before invading the cell.

Once inside, asexual reproduction begins. "Depending on the species, this reproduction can vary from two cycles to four cycles. One parasite can result in hundreds of thousands of copies", explains Urara Kawazoe, associate professor in the Department of Parasitology, and responsible for the development at the unit of the parasite strains that served in the experiments. In each of these cycles, a legion of parasites already reproduced in geometric progression breaks through the intestinal wall cell and moves to other cells in the intestine. Some species cause blood vessels to burst in and out of healthy cells, causing hemorrhage in birds. This is when the chicken's ability to gain weight is compromised. The nutrients present in the feed ingested by the chicken are not absorbed by the body, resulting in final weight loss and the generation of animals with low commercial value.

The last cycle of reproduction is carried out sexually, which results in the final forms of the protozoan, which will be excreted with feces. Before leaving the animal, Eimeria gains double encapsulation, more resistant to the external environment. It is this form that will allow the parasite to mature before re-infecting a healthy chicken that ingests the material. "The maturation of this, outside the animals' organism, occurs in 48 hours and it is necessary to have oxygen, light and a temperature between 25 and 30 degrees", explains Urara. The protozoan only causes damage to the chicken's body when ingested in mature form.

In vitro - In in vitro research it was discovered that the peptide can rupture the membrane of the first infective form of the parasite, the sporozoite. According to Silva Júnior, without this protection, Eimeria can no longer trigger the process of adhesion to the intestinal wall and the consequent entry of the parasite into the most favorable point for organizing asexual reproduction, triggering the multiplication of the parasite. As a result, the bird may even become infected, but it will not develop the disease and the body's ability to retain nutrients from the feed will not be interrupted, a key process for obtaining a commercial weight of 1,5 to 2 kilos within 45 days. .

There are other advantages of the amino acid sequence (peptide) created in research developed at Unicamp for application in poultry farming. In addition to controlling the disease, the product does not leave traces of foreign substances in the meat.

The combination of amino acids is absorbed by the body as protein, leaving no substance that can be traced later. This is a condition for access to important markets, mainly European and Asian. PW2 - the scientific baptism of the discovery - also contains two amino acids necessary for chicken development, Lysine and Tryptophan. These two essential amino acids are served to the flock as a dietary supplement. The supply of the peptide synthesized in the laboratory can, therefore, meet the demand for these amino acids and, in addition, protect the bird against coccidiosis.

New steps - To achieve this, the research will enter a fundamental phase - the massive production of the synthesized substance for use in commercial birds. According to Silva Júnior, negotiations with some pharmaceutical companies are trying to facilitate an agreement for the production of the peptide on a large scale. Among the options for producing this substance is the inclusion of the PW2 peptide coding sequence in corn DNA, which would require the production of genetically modified corn.

As Brazilian legislation for transgenics has become restrictive, the alternative has become to license the discovery to international pharmaceutical industries, in countries where development is permitted. The worldwide interest in alternative treatment of avian coccidiosis is great. It is estimated that the cost of commercial losses or disease control worldwide reaches around US$1,5 billion per year.

From the technique - If the result of the research in itself means an innovation (considering the unprecedented way of combating the disease without the use of medicines), the path traced by the researchers, coordinated by professor Adilson Leite - who passed away at the beginning of this year -, is also be unprecedented. The phage display technique for obtaining the ideal peptides intended for the task of attacking Eimeria sporozoites (the first phase of the parasite in the bird's intestine) had never been used for this purpose. To give you an idea of ​​what this means, the technique was used to develop one of the drugs in the anti-AIDS cocktail.

Much of the research, explains Silva Júnior, required time to identify in a library of peptides those with the appropriate "fit" for adhesion to the pathological agent's membrane. To achieve this, they used a virus that infects the bacteria Escherichia coli (type of virus called M13 bacteriophage). In one of the proteins expressed in the virus there were all the combinations of amino acids necessary to initiate a scan, capable of, in the final phase, resulting in a combination of 12 amino acids with the power to operate as an antimicrobial agent. "In fact, what we discovered here was something that opens up a huge range of possibilities for biotechnological use", explains Silva Júnior.

From this stage onwards, the research had the help of the National Center for Nuclear Magnetic Resonance at the Federal University of Rio de Janeiro (UFRJ). The team coordinated by researcher Fábio Almeida's group determined the three-dimensional structure of the peptide molecule designed at Unicamp.

The discovery has already been protected. A patent application has already been filed in the United States, Europe and Brazil. The financing for this was released by the Technology Patenting and Licensing Center (Nuplitec), a Fapesp organization. The patent secures the rights to the peptide, its variables and the method used to identify it.

Researcher develops live vaccine

The Department of Parasitology, at the Institute of Biology (IB) at Unicamp, is developing a Brazilian version of a live vaccine to combat avian coccidiosis. All The versions of vaccines of this type administered in Brazil are imported. The research consists of developing strains of Eimeria, the parasite that causes the disease with its shortened life cycle. The research will cover three of the seven species of existing protozoa. The selection criteria observed the most frequent and important species on commercial farms in Brazil, Eimeria acervulina, E. maxima and E. tenella. According to researcher Urara Kawazoe, the work has entered the final phase, with the development of the strain of E. tenella, one of the species of the genus Eimeria that produces hemorrhage when it attacks the chicken's intestinal cells. "This stage of work should last around two years", says Urara. With the premature development of these protozoan colonies, it is possible to create an attenuated vaccine, through which the chicken becomes infected, developing the parasite, but with minimal commercial damage, producing protective immunity in the birds. The shortening of the parasite cycle can reach 20% of the normal time, with variations depending on the species. The Eimeria protozoan with an early development cycle could, with the completion of the research, become the first live vaccine using this technique developed in Brazil. The version administered today to part of the Brazilian squad is made with an imported vaccine. This is not the only form of vaccination. A second, more used one, called virulent, also has an important place in the Brazilian market. It consists of spraying a small amount of the final form of the parasite onto the chicken. The objective in this case is to cause the animal's immune system to produce antibodies against Eimeria, allowing partially protective immunity against new infections. In this case, there may be a problem if vaccination management is not carried out properly. “The wrong dosage can produce the disease in the chicken instead of immunizing it”, explains Urara.

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