Heranza Equipamentos para Laboratórios
Ribeirão Preto company is developing a molecular diagnostic
test for disease endemic to the region transmitted to dogs by ticks

Lívia Komar

The area is small — 25 sq. meters (270 sq. ft) —, but the space is extremely organized. In the room of the Department of Biotechnology (Departamento de Biotecnologia) of the Universidade de Ribeirão Preto (University of Ribeirão Preto, Unaerp), four young people, three of them still undergraduate students and one just graduated, wearing white jackets, handle pipettes amid sophisticated equipment. Their goal is to develop a technique for the quick, cheap, precise diagnostic of a disease that is endemic to the region: ehrlichiosis. The name derives from the bacteria Ehrlichia canis, which infects ticks and, through them, dogs and other animals. The group works under the direction of the biologist Mozart de Azevedo Marins, a PhD in molecular biology who has decided to put his knowledge in the service of the diagnostic of that disease and other veterinary diseases common in the region.

To do that Marins created Heranza Equipamentos para Laboratórios (Heranza Equipment for Laboratories), a company that is still so small that it fits the room where it is located and its founder’s computer. With firm voice he didactically tells that the company was born in 2003 with the aim of solving local deficiencies. “I looked for problems in the region for which molecular biology could be useful. Talking with veterinarians I learned of diseases that had imprecise diagnoses, of which not even the infectious agents were known for sure. One of them was ehrlichiosis,” he recalls. Since then the investigation for an efficient diagnostic exam for the illness that affects the dogs of Ribeirão Preto – population 500,000, the prosperous center of Brazil’s richest sugar-producing region – has been Heranza’s main activity. Because it is transmitted by ticks, the disease is found mostly in tropical climate areas. The region’s typical hot climate is ideal for the reproduction of that acarus, which is common even in its urban areas. Humans may also be infected by the disease, but so far only one case, in Venezuela, has been reported in the medical literature.

The path Heranza has chosen to develop the exams is based on the analysis of the DNA of the animal believed to be infected through the Polymerase Chain Reaction (PCR) method. DNA identification through PCR is a common technique. Many laboratories diagnose ehrlichiosis through PCR; but in Ribeirão Preto, where the incidence is high, Heranza is pioneer.

Doing research with PIPE

For Marins, Heranza’s future is linked to the Programa Inovação Tecnológica em Pequenas Empresas (Technological Innovation in Small Businesses Program, PIPE), of the Fundação de Amparo à Pesquisa do Estado de São Paulo (State of São Paulo Research Foundation, Fapesp), whose funding the company has been getting since October of 2005, with the approval of the project "Análise da biodiversidade de bactérias associadas à erliquiose canina na região de Ribeirão Preto" (Analysis of the biodiversity of the bacteria associated to canine ehrlichiosis in the Ribeirão Preto region). There would be no other source to fund this research,” believes Heranza’s owner. “It’s very costly and the results are slow to come,” he explains. In addition to developing the diagnostic exam, the company has committed itself to monitoring the city’s dog population and the incidence of the diseases that affect these animals. The figures of the local Centro de Controle de Zoonoses (Center of Animal Disease Control) aren’t reliable — precisely for the lack of a precise diagnostic method. Heranza has already received nearly US$ 100,000 for purchasing equipment and material such as reagents. The four members of the company’s staff who work in the room where the company currently operates have fellowships also financed by the program.

But as of 2008 Marins wants Heranza to have its own headquarters, with profits coming from a target clientele: veterinary clinics. “With time, veterinarians will have to resort to more specific exams,” he believes. “Illnesses such as that are emerging all over the world, and the diseases transmitted by ticks are increasing ate an alarming rate,” he continues. That means more opportunities and a larger market for the still small innovating company.

Here’s a fact: according to the Brazilian Union of the Animal Health Products Industry (Sindicato Nacional da Indústria de Produtos para Saúde Animal, Sindan), the segment grew 7% in 2006 in Brazil, and closed the year with total sales of approximately US$ 1.2 billion. According to the entity, the positive numbers are the result of a growing concern of raisers with the health of their animals – they are investing increasingly more to reduce the risk of diseases. Another factor to be taken into consideration is the modernization of the Brazilian veterinary products manufacturing area, today one of the most advanced in the world, which today is capable of not only responding to Brazil’s sanitary demands, but also to the demands of other countries. For 2007, Sindan estimates a slightly smaller, but still significant growth of 3% to 4%.

The tests available

The first cases of ehrlichiosis were diagnosed in Brazil in 1973, through the clinical method. The symptoms are vomiting, fever, anemia, apathy and spontaneous bleedings. Later the blood platelet count, which indicates the animal’s reaction to an infection, but doesn’t identify the cause, began to be used for diagnoses. The so-called thrombocytopenia, however, can also be caused by other diseases with similar symptoms, such as, for instance, the one caused by the bacteria Babesia canis. “Because this is an endemic region, the veterinary associated the thrombocytopenia to ehrlichiosis and treated it erroneously with the antibiotic directed to that illness,” explains Marins. This imprecise method is still used today.

But in Ribeirão Preto the most commonly used test for the diagnosis of ehrlichiosis is the immunological – an alternative to the platelet count. This test provides answers about the antigens – viruses, bacteria and fungi cells – that may exist in the animal’s organism. The problem is that with this method often happens the so-called cross-reaction, that is, the substance that is found could be caused by other viruses and bacteria with similar characteristics present in the animal’s body.
With the use of the DNA analysis through PCR there is no uncertainty. Heranza develops its test using a PCR variation, called “nested PCR.” A sample of the animal’s blood is enough. If there’s an infection, together with the animal’s DNA will come up the identification of the bacteria’s DNA. The result is ready in 24 hours.

Phase II and new goals
In the project’s Phase II the company committed itself to expand its analyses to other parasites and hosts. Since 2006 Heranza has been working on the development of tests for horses, cattle and sheep, victims of several species of ticks that are disease vectors. For that the company has the collaboration of veterinarians, who provide free blood samples for the research.

One such collaborators is the veterinarian Gisele Nassif Conti, of Ribeirão Preto’s Nucleon Diagnósticos e Especialidades Veterinárias laboratory. She highlights the practicality and efficacy of the diagnostics through PCR, of 99.99%. “Only recently has biotechnology become part of the veterinary universe,” says Conti. “Many veterinarians aren’t familiar with diagnostic innovations and hold on to old methods, or to clinical methods alone. The cost of the exams is another problem. Animal owners are not used to the advances of the veterinary medicine. Information and clarification are keys. Veterinary is already a preventive medicine, not just curative medicine,” she concludes. Nucleon, where she works, collaborates with Heranza’s research: the clinic uses the company’s test in the animals at the price cost, of approximately US$ 28. Heranza claims that it still gets no commercial return with that.

Of the 700 dogs Heranza has analyzed since September of 2006 as part of the project financed by PIPE, 38.9% were infected with the Ehrlichia canis. The sick animals took antibiotics and their ticks were rigidly attacked. The remaining dogs were healthy or had the Babesia canis. In horses, cattle and sheep were found a number of microorganisms that cause pathologies transmitted by ticks: Babesia bovis, B. bigemina e Anaplasma marginale.

Marins explains that another advantage of a precise diagnostic is the animal’s well-being. “The treatment with antibiotics (doxycycline) costs between US$ 25 and US$ 34. The question, however, is not just the expenses with the antibiotic, but rather whether an antibiotic should be used or not. With more precise exams the veterinary is better equipped to prescribe the treatment and also to define when it’s time to stop it.”

Ideas that are multiplied with PIPE

The project also has the goal of stimulating knowledge through practice, providing specialization to the future biotechnologists who are working daily in the research associated with Heranza’s work at the university. “Here at the lab we learn in practice everything related to molecular biology. The techniques of analysis of sequences, extraction and cloning improve our knowledge for the future,” says Giovana Pirolla Cardozo, a scientific initiation student of the last year of the course of biotechnology who has been working at Heranza’s laboratory since August of 2006.

PIPE was a lever for the multiplication of the ideas involving Heranza’s future. There are many goals for the company’s growth. “PIPE’s work was one of detection. Now we want to migrate to a project to discover new molecules to combat the diseases,” hopes Marins.
Heranza plans to produce an exam kit for laboratories with all the reagents necessary for the identification of the Ehrlichia canis, the Babesia spp and the Anaplasma platys. The laboratory’s investment would be just the purchase of an equipment called thermocycler, which would make this diagnostic method cheaper and more popular among veterinarians.

Another side effect of Heranza’s work has been to stimulate the area of biocomputing at Unaerp. “One of my students has a Master’s Degree project for the development of a management software for our laboratory, from data storage to cataloguing, recuperation and even analysis,” says Marins. In his company’s “virtual headquarters” – his computer – he shows the program’s project, with the entire organogram of the procedures, from the collection of animal blood to the incidence of the disease in each area of the city.