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veterinary medicine

Thick shell

Vaccine protects birds from bacteria that causes fragility in their eggs

LIVET Bacteria attack the birds’ bone marrow and alter the calcification of the shellsLIVET

In 1991, the year was atypical for several farms in the interior of São Paulo. The hens laid an excessive number of eggs without a shell or with an extremely thin shell. It was something that called the attention of João Takashi Ohashi, a veterinarian graduated from the University of São Paulo, who a short time previously had set up his own company, Livet Produtos Veterinários, after having worked for a long time on the development of products in a multinational company. He decided to look into the causes of this strange occurrence, drawing up a hypothesis that part of the problem could be connected with infectious agents like bacteria.

Until then, studies indicated old age in the hens, poor feeding, and genetic and environmental problems as factors responsible for alterations in eggshells and specific infectious diseases. Years later, João went ahead with his idea of investigating the bacterial causes of the disease, and went so far as to imagine a vaccine. In 1998, to make his studies a reality, he presented a project to FAPESP, under the Small Business Innovation Research (PIPE) program.

The project was coordinated by Professor Masaio Mizuno Ishizuka, from USP’s School of Veterinary Medicine and Zootechny. “Concluded in June last year, the researches showed a correlation between lesions in the spinal cord caused by bacteria and the quality of the shell,” says João. And they resulted in an unprecedented bacterial vaccine, effective in eliminating 11% of the problem of malformation of the shell, according to Masaio.

Furthermore, the studies indicated a virus as being responsible for much of the damage to the metabolism of calcium in the birds. Added together, these two causes represent half of the problems related to ovosporosis, baptized by the researchers as osteoporosis in birds. The remaining 50% refers to factors like inadequate nutrition and handling, and diseases that cause lesions of the reproductive tract, bronchitis and other infections. At the moment, João and Masaio are poring over the development of a mixed vaccine (bacterial and viral) to minimize the losses resulting from the poor quality of eggshells. These losses reach 7.4% a year in Brazil, 6.4% in the United States, and 8% in Germany.

A survey by the Brazilian Institute of Geography and Statistics (IBGE), published at the beginning of this year, involving only poultry farms with a stock of 10,000 laying hens or more, shows that Brazil produced 1.332 billion dozens of eggs, or about 16 billion individual eggs, in the period from January to September 2002. The forecast for this year amounts to around 21.3 billion eggs. This volume gives an idea of the dimension of the losses faced by the sector in discarding eggs improper for consumption. Considering a country that wants to attain zero hunger, the losses are immense, possibly amounting to 1.3 billion eggs lost in 2002.

Progressive disease
The vaccine developed by Livet is intended to fight Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus sp. bacteria in isolated lineages of hens. The study indicated that these bacteria reach the birds’ spinal cord, preventing the proper formation of calcium in the eggs. Vaccination should be carried out by the 16th week of the birds’ lives, when the reproductive stage begins for laying hens. “In the beginning, the disease causes small losses, which are intensified as the bird comes to the end of the reproductive stage, reaching 15%, because it is a chronic and progressive disease,” Masaio reports. “Ovosporosis begins much before the production of eggs begins.”

The studies began with the isolation and identification of the bacteria in the bone marrow of the femur and the tibia of birds with ages between 1 day and 16 weeks, selected at random in the sheds of four commercial farms that showed problems with losses related to the low quality of the shells. The poultry farms were similar regarding their size, the kind of food and the zootechnical and sanitary handling.

Degrading toxins
To stimulate production and losses deriving from the poor quality of the shells, birds at the productive stage from other sheds at the same farms were selected, with ages corresponding to 20, 40, 60 and 80 weeks of life. The experiments revealed a relation between bacterial infection in bone marrow and intensification of calcification in the marrow. “The more serious the lesion, the lower the quantity of osteoblasts and osteoclasts, the cells of the bone marrow,” João reports. The osteoblasts are responsible for synthesizing the organic part of the osseous matrix, while the osteoclasts are linked to the reallocation of calcium in the bone tissue.

According to the researcher, the bacteria produce toxins capable of degrading parathyroid hormone (PTH), a hormone responsible for controlling the quantity of calcium released by the bone marrow for the formation of the eggshell. The hypothesis that infectious agent could be part of the problem was corroborated by means of bacteriological, histopathological (a microscopic study of organic lesions) and statistical tests. After this, the researchers worked on the development of a vaccine to control the bacteria. Tests carried out in the laboratory showed that this vaccine increased by 11% the number of eggs with a good shell, that is, in a good condition for being marketed, a promising result when one is dealing with a disease with many causes.

Test of the ring
But the result did not totally please the researchers. They then decided to broaden the subject of the study and began trying to identify viruses that could influence the birds’ metabolism of calcium and, as a consequence, the formation of the shell. The process for identifying and isolating the virus was carried out by a method for diagnosis called Bi-Digital O-Ring Test, created by Japanese researcher Yoshiaki Omura, who lives in the United States, and which is mainly used by acupuncture physicians to investigate the influence of electromagnetic energy on the diagnosis and treatment of diseases.

The test is based on resonance, a principle of physics, which is the prolongation of a sound or electromagnetic wave by its reflection or repercussion on other bodies. João says that with this method he manages to identify, among other things, grains of several cereals wrapped up in black plastic. He compares the diagnosis to the work of a pointer dog that sniffs out the prey. “With O-Ring, I go directly to what interests me, both for identifying the viruses and bacteria and for developing the products, which include phytotherapics for treating cattle and poultry.” The confirmation is always done by the conventional technique, as in the case of the virus that is being analyzed and typified by the Brazilian Agricultural Research Corporation (Embrapa).

As soon as confirmation takes place, which the researchers expect to happen before the end of February, they will begin to test, in the laboratory, the vaccine to fight the virus. The next step is to go in with the request for patenting the bacterial vaccine, in Brazil to start with, and then abroad. The registration of the viral version will be left for a later stage, after the tests have been concluded.

Calculations made by American producers indicate that the losses in the United States, solely as a result of the malformation of the shells, are in the order of US$ 1.2 per bird a year. A survey carried out in 1997 indicates that in the immediately preceding year this problem caused Brazilian producers losses that amounted to R$ 79 million. A proof that the vaccine, as soon as it is completely ready to prevent the viruses and bacteria from interfering with the birds’ metabolism of calcium, will mean considerable savings for the Brazilian poultry raising sector.

The Project
Development of a Bacterial-Toxoid Vaccine for Preventing the Syndrome of Poor Quality Eggshells in Reproducing and Laying Commercial Poultry (nº 98/14865-1); Modality Small Business Innovation Research (PIPE) Program; Coordinator Masaio Mizuno Ishizuka – USP/Livet; Investment R$ 237,824.50 and US$ 17,211.00

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