Darwin Chronicles

May 2006

Marburg viral vaccine prevented symptoms in animal studies

An experimental vaccine against Marburg virus, an African Filovirus, appears efficacious in nonhuman primates, when given after they are exposed to the virus, according to a study.

Marburg hemorrhagic fever is an uncommon, but often fatal, disease, with case fatality rates of about 90%. There is no effective way to prevent or treat the virus once someone is infected, which causes great concern for researchers developing countermeasures against potential bioterrorist threats.

U.S. and Canadian researchers have developed a vaccine from an attenuated recombinant vesicular stomatitis virus (rVSV). They administered the rVSV vaccine to five rhesus macaques, aged 4 to 6 years, 20 to 30 minutes after exposure to a high dose of Marburg virus. Three other macaques acted as controls. Three of the five vaccinated animals developed a fever by day 6, but their temperature returned to normal by day 10, and all of them survived.

All three control monkeys that were not protected with the vaccine died within two weeks. This result demonstrated that it might be possible to use rVSV vaccines to treat Marburg and similar viruses, such as Ebola, after infection, researchers said. Studies are now in progress to determine how late after exposure these vaccines might be beneficial.

In related news, researchers at the Greene Infectious Disease Laboratory at Columbia University Mailman School of Public Health have developed a rapid, comprehensive diagnostic test for viral hemorrhagic fevers caused by the Ebola and Marburg viruses, as well as others (Emerg Infect Dis [serial online]. April 2006. Available at: www.cdc.gov/ncidod/eid/vol12no04/05-1515.htm).

“Currently, there is no way to treat most of these outbreaks,” W. Ian Lipkin, MD, director of the Jerome L. and Dawn Greene Infectious Disease Laboratory and professor of epidemiology, neurology and pathology at Columbia University, said in a release. “The most important first step is diagnostic — rapid identification of the exact pathogen responsible for an outbreak of disease is critical for containment and the implementation of public health measures, especially in instances where the agents are encountered out of their natural geographic context.”

Although other tools exist for the detection of viral hemorrhagic fever (VHF) agents, none offers the sensitivity and speed of this new diagnostic screen, which incorporates MassTag polymerase chain reaction technology, which can simultaneously consider multiple agents.

To facilitate rapid differential diagnosis of VHF agents, Briese and colleagues established the “Greene MassTag Panel VHF v1.0,” which can screen simultaneously for Ebola Zaire, Ebola Sudan, Marburg, Lassa virus, Rift Valley fever, Crimean-Congo hemorrhagic fever, Hantaan, Seoul, yellow fever and Kyasanur Forest disease virus.

Scientists design potent anthrax toxin inhibitor

Scientists have engineered a powerful inhibitor of anthrax toxin that works well in small-scale animal tests.

Ravi S. Kane, PhD, of Rensselaer Polytechnic Institute in Troy, N.Y., and Jeremy Mogridge, PhD, of the University of Toronto, and colleagues built a fatty bubble studded with small proteins that can cling tightly to the cell membrane receptor-binding protein used by anthrax toxin to gain entry into a host cell (Nat Biotechnol. April 23, 2006).

The protein-spiked fatty bubble, or functionalized liposome, hampers a critical early step in the assembly process that anthrax toxin must undergo to become fully active. In vitro, the inhibitor, which is covered with multiple peptides, was 10,000 times more potent than unattached peptides.

Bacillus anthracis produces a toxin that causes anthrax symptoms. Antibiotics are used to treat anthrax, but even with such therapy, inhalation anthrax, the most severe form of the disease, has a fatality rate of 75%.

Anthrax toxin has three parts: protective antigen, a protein that binds to a receptor on the target cell surface, and two enzymes that must be transported into the cell to cause damage. The enzymatic portions of the toxin enter the cell through a pore created for them by the protective antigen after it binds to the cell’s outer surface. The protective antigen can be seen as a bundle of seven cigar-shaped parts, a molecular arrangement referred to as “polyvalent.”

The inhibitor designed by Kane and colleagues is also polyvalent. The polyvalent inhibitor binds the toxin at multiple sites and is more potent than an inhibitor that binds at a single site. The multiple peptides on the functionalized liposome are arranged with the same average spacing as the binding sites of the protective antigen molecule, which permits a firmer bond between the two, explains Kane. When the inhibitor is bound tightly to the protective antigen, the subsequent steps of enzyme entry cannot occur and the toxin is effectively neutralized.

The investigators tested the anthrax inhibitor in rats. Injection of the inhibitor in relatively small doses at the same time as anthrax toxin prevented five out of nine rats from becoming ill. Slightly higher doses of the inhibitor prevented eight out of nine rats from becoming ill. Kane and colleagues injected nine additional rats with anthrax toxin only. Of these, eight became gravely ill. This experiment was the first to show the efficacy of a liposome-based polyvalent inhibitor in animals, according to Kane.

In a release, Kane said the recent experiments demonstrate a proof of principle and suggest that polyvalent inhibitors can be used along with antibiotics in a clinical setting. Aside from its inherent toxicity, anthrax toxin also accelerates the disease process. Thus, combining antibiotics with a toxin inhibitor may act synergistically to lessen or halt anthrax symptoms.

Using the same technique of placing multiple peptides on a liposome, the researchers created a polyvalent inhibitor of cholera toxin that functioned well in test-tube experiments.

Restricting antibiotic use lowers resistance levels

Australia’s policy of restricting antibiotic use in food-producing animals may be linked with lower levels of drug-resistant bacteria in humans, according to an article in Clinical Infectious Diseases.

Campylobacter jejuni is a leading bacterial cause of foodborne illnesses in industrialized countries, but drug resistance has made many Campylobacter infections difficult for physicians to treat. This means longer bouts of diarrhea and a higher risk of serious or fatal illness. Bacterial resistance to drugs is generally attributed to inappropriate prescribing or overusing antibiotics.

An Australian solution to the drug resistance problem has been to prohibit the use of the fluoroquinolone class of drugs in food animals, such as poultry. Afterward, Australian researchers examined C. jejuni isolates that they collected from 585 patients in five Australian states. None of the patients had received fluoroquinolones within the month prior to becoming ill. The researchers discovered that only 2% of the locally acquired Campylobacter isolates were resistant to ciprofloxacin. Countries that allow fluoroquinolone use in animals may have a drug resistance prevalence of up to 29%.

“There are different causes that lead to bacterial antibiotic resistance, and use of antibiotics in food animals is only one of the multiple causes,” said Leanne Unicomb, epidemiologist with OzFoodNet and Australia National University. However, the evidence indicated that “use of fluoroquinolones in food animals in other countries has increased the risk of resistance in [Campylobacter] isolates infecting humans,” she said in a release. The researchers concluded that the low drug resistance they found “probably reflects Australia’s policy of prohibiting fluoroquinolones for animal use.”

Norway and Sweden, with similar restrictions in place, have also reported low resistance rates. The United States took the cue and banned fluoroquinolones in poultry in September 2005.

Curtailing wildlife trade would go long way toward eliminating zoonoses

As millions of animals are traded across borders each year, and much of this trade is illegal and undocumented, it is not surprising to learn that this trade is a leading cause of zoonotic diseases, said Robert Cook, VMD, MPA, at the International Conference for Emerging Diseases meeting.

“As you move animals around the world into these market places where sanitation and hygiene are a big question mark, you create possibilities for disease mutation and jumps. So it is not really surprising that we have been seeing the numbers of diseases, what is surprising is that we are not seeing more of them,” said Cook, who is chief veterinarian and vice president of wild life health for the Wildlife Conservation Society (WCS).

“The movement of people has sped up dramatically as the population has increased and technology has improved so we can pretty much move anything around the planet in 24 hours or less,” Cook said.

Animal trade

The World Trade Organization estimated that the legitimate global trade in agricultural products was $783 billion in 2004. “The movement of agricultural products is fairly well defined throughout the world. It is not the same for the wildlife trade, a trade that is totally undefined because much of it is illegal.

“We do know that tens of millions of wild animals are being shipped out of the forest every year for food, for pets and for traditional medicine,” he said. “This is quite alarming, because we are only at the edge of what will be a dramatic increase in the global demand for meat protein over the next 20 years.”

Three hundred fifty million live plants and animals are shipped globally each year, according to Cook, and of those approximately 40,000 live primates, 4 million birds and 640,000 live reptiles are shipped, which has large implications for public health, because each species has been traced to myriad infections. One CDC study looked at one aspect of this trade: the live reptiles imported into the United States between 1994 and 1995. More than 170,000 reptiles were imported into the United States in that period, and more than 54,000 had ticks, according to Cook.

In much of the developing world, this trade is done in open marketplaces, where hundreds of thousands of live and butchered exotic animals from armadillo to non-human primates are bought and sold. In China, these live animal markets played a role in the SARS epidemic, when civet cats were implicated as the source. In 1997, in Hong Kong millions of birds were culled from these markets in an attempt to stop avian influenza.

The numbers of animals that move through these markets is phenomenal. For example, one Thai market sold 70,000 birds of 276 species over 25 weekends that were observed by researchers. In Central Africa 1 billion kg per year of wild meat is traded for local and regional consumption.

As lands are deforested, people in remote areas can move more easily from the forest to the urban areas, bringing with them the animals they’ve hunted. Often these are non-human primates. Non-human primate meat is not first on most people’s list of preferred foods, but it is often the easiest to hunt, according to Cook, and many of these cultures depend on hunting for food. “Getting today’s meal means doing it as quickly as possible and getting back home and that has implications because of a variety of reasons.”

Better surveillance

Better surveillance and training are needed. However, there are programs addressing this issue, according to Cook. For instance, in Gabon, large numbers of non-human primates were dying, and there were indications that they had succumbed to Ebola. A veterinary team of local Gabonese trained and accompanied by WCS field veterinarians investigated and found gorillas, chimpanzees and other non-human primates were dead. Some did test positive for Ebola. Epidemiological investigations since that time suggest that bats may be the reservoir for this disease, according to Cook. One of the important aspects of this investigation was the role that locals played in the investigation.

“The Gabonese government primate research facility took the lead on the bat research. The last five outbreaks of Ebola virus in this region of the world are believed to be linked to bush meat – bringing out sick or dead non-human primates that people then eat, and the disease begins to pass from person to person,” Cook said.

The real key here is teaching people about the potential threats. It is less about the reaction to the disease and being more proactive prior to the disease so that they understand the potential threat to their environment. And the message here was that sick or dead primates are a potential danger to people.

A big concern right now is avian influenza, and whether it will become a human pandemic. Everyone is watching the migratory flyways of wild birds. Cook said they should include surveillance of the wild animal trade.

“As you’re pointing to the sky and saying you know it’s that migratory bird, no one is paying any attention to the 747 filled with products,” he said, adding that officials need to focus on the illegal animals crossing the borders. “It is not migratory birds or the trade; it is migratory birds and the trade,” he said.

He sited one known example, where a man tried to import Mountain Hawk eagles from Thailand into Belgium; the animals tested positive for H5N1 avian influenza.

“This kind of illegal trade is going on all the time, we don’t catch most of it and that is what is scary about this. We need to think much more seriously about surveillance upstream.”

Stopping the illegal trading

Cook cited several steps that might help decrease the illegal trade in wild animals, which could decrease the importation of zoonotic diseases, such as monkeypox.

Improve surveillance of U.S. ports, and help other governments improve surveillance upstream where these diseases are likely to originate.

Curtail the trade in wildlife. “If we could do that we would all be a lot better protected,” he said.

Improve the way animals are handled in live markets and decrease the sale of wildlife for food and products, as well as improve the sanitation in live animal markets in many parts of the world.

“Improving those conditions will go a long way in protecting us all,” Cook said. — Marie Rosenthal