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Genetic therapy for hens

Unicamp researchers discover protein for fighting bird disease

URARA KAWAZOE / UNICAMPWhen it catches coccidiosis, the chicken gets an intestinal infection and loses weight URARA KAWAZOE / UNICAMP

Amid so many possibilities offered by biotechnology, researchers from the Molecular Biology and Genetic Engineering Center (CBMEG) at the State University of Campinas (Unicamp) have succeeded in taking a great step forward for formulating a new medicine for the treatment of coccidiosis in chickens, a disease that causes an infection in the intestines, debility and loss of weigh in poultry. In a small protein (peptide), they identified the way towards a new therapy for fighting the protozoa (Eimeria spp.) that are responsible for the disease. The extent of this discovery lies in the annual losses brought about by coccidiosis to Brazilian poultry breeders: US$ 20 million. Part of this loss comes from today’s anticoccidian medicines, which are losing the war against this protozoon, generating individuals that are resistant to the drugs.

Under the coordination of Professor Adilson Leite, the researchers’ original idea is to create a variety of transgenic corn incorporating the peptide in its seeds and to make it part of the basic poultry feed. This treatment has now awoken the interest of two multinational companies – one from America and the other from Canada -, which are analyzing the possibility of going ahead with the studies. The researchers’ knowledge is safeguarded by a patent request filed both in the United States and Europe and in Brazil. Financed by FAPESP, through the Technology Patenting and Licensing Nucleus (Nuplitec), the patent is valid for the peptide itself, for its variants, and for the identification method as well.

For Leite, one of the secrets of success was studying in detail the cycle of the development of the protozoon called Eimeria acervulina, one of the seven species that cause coccidiosis, until defining the stage most vulnerable to possible treatments. For this work, the team from the CBMEG had the collaboration of Professor Urara Kawazoe, who is responsible for the Chicken Coccidiosis Laboratory of the Parasitology Department of Unicamp’s Institute of Biology (IB). The researchers knew that the transmission of the disease occurs when the hens scratch the ground and swallow the oocysts (an “egg” enclosed by two resistant protective covers) of Eimeria eliminated together with the feces of diseased birds.

Invasion by the parasite
In the animal’s gizzard, after being swallowed, the oocyst is broken, and the first cover bursts. Right after that, on account of the digestive enzymes, the invasive forms of the parasite, called sporozoites, are released from the second cover. This happens in the bird’s intestine, after passing through the stomach. In the final stage, the parasite first sticks to and then invades the cells of the inside surface of the intestines, using them as a new shield against the animals’ immune system.

“The stage in which the parasite is most vulnerable is at the moment when the sporozoites are released. Then, there is no protection. This was the point at which we had to act”, explains Arnaldo da Silva Junior, who is also a researcher at the CBMEG. When they disembark in the intestine, the Eimeria sporozoites recognize the place and secrete adhesive proteins. If they could succeed in identifying peptides that could block the action of these recognizing or adherent proteins, the group from Unicamp would prevent the invasion of the cells and would take a great step towards a new possibility of treating the disease.

To find the possible fighting peptides, the team opted for a selection method called phage display. They used a virus that infects the Escherichia coli bacterium – called bacteriophage m13 – which worked like a library of peptides. In one of its proteins, the organism showed peptides that involved all the combinations and arrangements of sequences that the researchers needed. “There were 20 amino acids in 12 different positions, which generates an astronomical amount of information. All we needed was there”, warrants Silva Junior.

In the laboratory, the sporozoites were repeatedly put in contact with the bacteriophages m13. After this process, those viruses containing peptides that showed affinity with the Eimeria sporozoites were separated and identified. “They were the best of the best”, says Silva Junior. Finally, the researchers noticed that the selection converged on a single peptide that, when tested, showed anticoccidian activity. Called PW2, it was chemically synthesized and, always in the laboratory, incubated with the parasites, to test its effectiveness. The graphic representation of the three-dimensional structure of the molecule, which will allow future studies for the new medicine to be developed, was built by the National Magnetic Resonance Center of the Federal University of Rio de Janeiro (UFRJ).

In small concentrations, PW2 successfully blocks by up to 70% the invasion of the cells in the chickens’ intestines by the protozoa. Better still: it was found that the peptide sets off alterations in the permeability of the parasite membrane, which leads the researchers to work on the hypothesis that, even if the remaining 30% manage to reach the surface of the birds’ intestines, they will probably fail when they try to carry on with their cycle. In the course of the studies, the PW2 was tested against two species of the protozoon, which are the most frequent ones in Brazil: Eimeria acervulina and Eimeria tenella. There are also another five kinds, which were not assessed, since they are not significant. But the researchers believe that the beneficial effect will be the same.

Gene in the corn
The new stages of the studies are tests with live birds and the creation of corn that brings in its genetic code the PW2 anticoccidian peptide. The technology for obtaining this variety is known of old to the group from Unicamp (see Pesquisa FAPESP nº 49). “You only have to introduce an expression cassette into the seed of the grain, containing the gene in question”, Leite sums up. The idea brings with it another great advantage: to boot and unwittingly, the researchers found that the peptide discovered is also effective in inhibiting the action of a few fungi, including the ones of the Aspergillus genus, precisely those that cause mildew in stored corn. “We will be winning at both ends”, concludes the coordinator of the studies.

The novelties are coming at the right moment. The incidence of chicken coccidiosis in Brazil is plotting a rising curve. After remaining stationary at less than 10% in the 80’s, because of medicines, it started to climb and reached the 40% mark in the 90%, as the protozoon became resistant to the medication in use. Coccidiosis a morbid disease, that is to say, in many cases, its signs are perceived only when the bird is slaughtered, when there is nothing else to be done.

It does not kill the chicken, but by setting off an intestinal infection, makes the animal lose the capacity for transforming the feed it consumes into weight. The discovery of PW2, may therefore represent a new stage in the history of the fight against chicken coccidiosis; it may even reduce costs in poultry breeding, since the preventative medicine could already be incorporated with the feed itself.

The project
Patenting request for a New Method for Selecting Antimicrobial Peptides and the Anticoccidian Peptide PW2 (nº 00/12151-3); Modality Intellectual Property Support Program; Coordinator Adilson Leite – Unicamp; Investment R$ 65,044.05