Constantine Alexander's blog

Antimicrobials from personal care products found in statewide survey of Minnesota's rivers and lakes

		Postdoctoral research associate Benny Pycke holds a sediment sample from a Minnesota lake. The samples were collected from the upper 10 cm of the bed surface and stored in a freezer prior to analysis. Credit: Biodesign Institute at Arizona State University

In our zest for cleanliness, have we permanently muddied our nation's waters?

A science team from Arizona State University, in collaboration with federal partners, has completed the first statewide analysis of freshwater bodies in Minnesota, finding widespread evidence of the presence of active ingredients of personal care products in Minnesota lakes, streams and rivers.

These products are a billion dollar industry and can be found in antimicrobial soaps, disinfectants, and sanitizers to scrub our hands and clean countertops. Hundreds of antimicrobial products are sold in the U.S., many marketed with efficacy claims that remain elusive due to the short duration of the average consumer's handwashing practices. The fate of these products can be traced from home use to sewers to wastewater treatment plants to eventually, downstream bodies of water.

The research team focused on two active ingredients found prominently in antibacterial soaps;triclosan and triclocarban; which have come under scrutiny by the EPA and FDA due to their environmental and human health concerns. These compounds persist for decades in the environment, and both triclocarban and triclosan are among the top ten pharmaceuticals and personal care products most frequently found in the environment and in U.S. drinking water resources.

"This study underscores the extent to which additives of antimicrobial consumer products are polluting freshwater environments in the U.S.; it also shows natural degradation processes to be too slow to counter the continuous environmental release of these endocrine disrupting chemicals," said Halden, director of Environmental Security at the Biodesign Institute and professor in the Ira A. Fulton School of Sustainable Engineering and the Built Environment. Halden's research focuses on the interconnectedness of the water cycle and human health, with special emphasis on the role of manmade products and human lifestyle choices on environmental quality.

In a previous study, Halden's team found significant concentrations of harmful soap related chemicals dating back to the 1950's in sediments of Jamaica Bay and Chesapeake Bay, into which New York City and Baltimore discharge their treated domestic wastewater, respectively.

		This is a close-up of the sediment sample. Credit: Biodesign Institute at Arizona State University

Upon their use, triclosan and triclocarban are absorbed through the skin and hence contaminate human blood, urine, and even breast milk. Ultimately, these chemicals together with the pharmaceuticals we use end up in our sewage and surface waters. In 2002, the USGS published a landmark study that showed 80 percent of 139 streams sampled from across 30 U.S. states were found to contain measurable levels of organic wastewater contaminants. The human health risks associated with these personal care product chemicals are still not fully understood despite them being used for decades.

In the ASU study, river, creek and lakebed sediment samples from 12 locations upstream and downstream of wastewater treatment plants were analyzed for the presence of antimicrobial compounds.

For Halden's team, which consisted of postdoctoral researcher Benny Pycke, environmental engineering graduate student and first author Arjun Venkatesan, the results showed that overall concentrations of triclocarban were 3- to 58-times higher than those of the more frequently monitored triclosan.

"We were able to detect these two compounds both upstream and downstream of suspected input sources, and the levels of the antimicrobial soap ingredient triclocarban were usually higher compared to triclosan," said Venkatesan. "Although triclosan is used in a larger number of formulations and personal care products, we found triclocarban to be more abundant in freshwater environments." The team also found degradation products of TCC but transformation of this antimicrobial is known to be very slow in natural environments.

"Also, we expected to find these compounds mostly downstream of wastewater treatment plants; but when we consistently found detectable levels upstream and downstream, we realized that there are probably multiple sources contributing to the contamination of these sites, potentially including additional wastewater treatment plants further upstream and runoff from sites where antimicrobial-laden sewage sludge had been applied."

		Benny Pycke and Fulton School graduate student Arjun Venkatesan look at data measuring the amount of personal care product active ingredients, triclocarban and triclosan, in the samples. Credit: Biodesign Institute at Arizona State University

"Every site is essentially downstream of something," added Pycke. A site in the immediate vicinity of a wastewater treatment plant near Duluth (St. Louis Bay at Lake Superior) had the greatest concentration of triclocarban and its lower chlorinated derivatives, and the Duluth site and Shagawa Lake site had concentrations three times higher than river and creek sediments. There was a strong correlation between the level of contamination with wastewater treatment plant discharge, stream flow and the population density of the surrounding region.

"As the name suggests, these antimicrobial compounds (triclosan and triclocarban) are incompatible with biological wastewater treatment infrastructure paid for with tax dollars," said Halden. "Municipalities in Minnesota and across the U.S. work hard using state-of-the-art equipment to keep our freshwater environments clean but they cannot control what consumers, misled by aggressive marketing, discharge into the sewage collection system."

Wherever antimicrobial personal care products are in use, water and sediment have been contaminated, a situation that certainly is not unique to the state of Minnesota. "Regulatory agencies are aware of the overuse of antimicrobials but no state or federal restrictions have been implemented yet for either triclosan or triclocarban," said Halden. "Aside from ecological concerns, widespread environmental occurrence of antimicrobials also is a potential public health concern because unwarranted use of antimicrobials can promote drug resistance of human pathogens."

Halden's research is developing engineering solutions to clean up environments impacted by antimicrobial compounds. However, he emphasizes that the best solution right now in combating this pollution is for consumers to limit their use of antimicrobial personal care products that, ironically, provide no measurable health benefits to the average consumer, as determined by an expert panel convened by the Food and Drug Administration in 2005.

For this project, ASU was supported by funding from the National Institute of Environmental Health Sciences. In addition to Halden's appointment as Director of Environmental Security at ASU's Biodesign Institute, he holds the title of professor in the School of Sustainable Engineering and the Built Environment, at the Ira. A. Fulton Schools of Engineering, ASU, and adjunct associate professor of Environmental Health Sciences, at the Johns Hopkins Bloomberg School of Public Health.

Suggested Readings:

• A.K. Venkatesan, et al., Occurrence of triclosan, triclocarban, and its lesser chlorinated congeners in Minnesota freshwater sediments collected near wastewater treatment plants, J. Hazard. Mater. (2012), Vol. 229-­230, Pages 29-35.  (accessed August, 2012).
• U.S. Environmental Protection Agency, St. Louis River Area of Concern, 546 2011.
• "Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations." Published by the American Chemical Society (ACS) in 2010.
• Daughton, C. G., Scuderi, M. S. T., Eds. Pharmaceuticals and Personal Care Products (PPCPs): Relevant Literature; U.S. Environmental Protection Agency: Las Vegas, NV, February 19, 2008. 
• An AP Investigation: Pharmaceuticals Found in Drinking Water, 2008. Associated Press. 

Contact: Joe Caspermeyer
joseph.caspermeyer@asu.edu
480-727-0369
Arizona State University

 

 

Giving to charity

Why do we donate more money to individuals when they are members of a group?

When charity recipients seem to belong to a cohesive group, donors will make stronger judgments about the victims, which leads to greater concern and increased donations if these judgments are positive, according to a new study in the Journal of Consumer Research.

"One of the most puzzling aspects of charitable giving is the relative meagerness of donations to large numbers of 'statistical' victims in contrast to the generosity shown to a single identified victim," write authors Robert W. Smith (University of Michigan), David Faro (London Business School), and Katherine A. Burson (University of Michigan). "A solution to this problem is to make multiple victims seem like a single, unified group."

The authors tracked funding activity on Kiva.org, a micro-financing website where lenders support groups of borrowers whose pictures are prominently displayed on the site. Independent ratings showed that some of the pictures on the website seemed to portray very tight groups while others rather loose groups. The authors found that groups that looked unified were more quickly funded. In other studies, the authors discovered that donations to help poor children were higher when the children were described as members of the same family.

However, the opposite is true when consumers are asked to donate to groups they may not view positively. Another study asked consumers to make donations to benefit child prisoners with poor living conditions. Those who read about the child prisoners that seemed unified had more negative judgments and gave them less money than those who read about the non-unified but otherwise identical child prisoners.

"Perceived group membership results in stronger judgments of victims. Victims are viewed more favorably when they belong to a group with positive traits, triggering greater feelings of concern and higher donations, whereas the opposite is true for victims sharing negative traits," the authors conclude.

Robert W. Smith, David Faro, and Katherine A. Burson. "More for the Many: The Influence of Entitativity on Charitable Giving." Journal of Consumer Research: February 2013.

Contact: Mary-Ann Twist
JCR@bus.wisc.edu
608-255-5582
University of Chicago Press Journals

 

 

Study proves that 1 extinction leads to another

		A parasitic wasp attacks an aphid in the first study to prove the theory that the extinction of one predator can lead to the extinction of another. Credit: Dirk Sanders

When a carnivore becomes extinct, other predatory species could soon follow, according to new research. Scientists have previously put forward this theory, but a University of Exeter team has now carried out the first experiment to prove it.

Published today (15 August 2012) in the Royal Society journal Biology Letters, the study shows how the demise of one carnivore species can indirectly cause another to become extinct. The University of Exeter team believes any extinction can create a ripple effect across a food web, with far-reaching consequences for many other animals.

The research adds weight to growing evidence that a 'single species' approach to conservation, for example in fisheries management, is misguided. Instead the focus needs to be holistic, encompassing species across an entire ecosystem.

The researchers bred two species of parasitic wasps, along with the two types of aphids on which each wasp exclusively feeds. They set up tanks with different combinations of the species and observed them for eight weeks. In tanks that did not include the first species of wasp, the second went extinct within a few generations. In tanks in which they co-existed, both wasp species thrived.

In the absence of the first wasp species, its prey grew in numbers. This threatened the other aphid, which the second wasp species attacks, eventually leading to its extinction. Both types of aphids feed on the same plants and there was not enough food for one to survive when the other thrived in the absence of its wasp predator.

Lead researcher Dr Frank van Veen of the University of Exeter's Centre for Ecology and Conservation said: "Our experiment provides the first proof of something that biologists have argued for a long time: predators can have indirect effects on each other, to the extent that when one species is lost, the loss of these indirect effects can lead to further extinctions. Although our study focused on insects, the principle would be the same for predators in any ecosystem, ranging from big cats on the African plains to fish in our seas.

"Our research highlights the fact that a 'single species' approach to conservation can be ineffective and even counter-productive. For example, protecting cod could lead to increased fishing pressure on other predatory fish which then, by the mechanism we have demonstrated here, could lead to further negative effects on the cod."

The experiment was designed by a team of University of Exeter scientists and second-year undergraduate students. The idea came about during a seminar, in which students were challenged to design an experiment that could prove the theory that predators have indirect effects on each other. The students were so inspired by the idea of proving a long-held theory that more than 30 of them volunteered to conduct the experiment with their lecturers.

Contact: Sarah Hoyle
s.hoyle@exeter.ac.uk
44-139-272-2062
University of Exeter

 

 

Widespread local extinctions in tropical forest 'remnants'

The small fragments of tropical forests left behind after deforestation are suffering extensive species extinction, according to new research led by the University of East Anglia (UEA).

Publishing today in the journal PLoS ONE, the researchers carried out a comprehensive assessment to estimate the long-term impact of forest fragmentation and hunting on tropical biodiversity in Brazil.

They studied the Atlantic Forest of eastern Brazil, including the region's largest and least disturbed old-growth forest remnants, and found that remaining habitat fragments had been virtually emptied of their forest wildlife.

White-lipped peccaries were completely wiped out, while jaguars, lowland tapirs, woolly spider-monkeys and giant anteaters were virtually extinct. Defaunation even extended to forest remnants with relatively intact canopy structures.

Widespread agricultural expansion has transformed the world's tropical forests, leaving few remaining blocks of primary forests unaltered by humans. There have been scattered reports of large mammal extinctions throughout Brazil, but the conservation value of a rapidly growing number of small forest remnants in highly-fragmented tropical forest landscapes has been hotly debated.

Senior author Prof Carlos Peres, of UEA's School of Environmental Sciences, said: "You might expect forest fragments with a relatively intact canopy structure to still support high levels of biodiversity. Our study demonstrates that this is rarely the case, unless these fragments are strictly protected from hunting pressure.

"There is no substitute for strict protection of remaining forest fragments in biodiversity hotspots like the Brazilian Atlantic Forest. Protection of forest cover alone is not enough to sustain tropical forest species, as overhunting compounds the detrimental effects of small habitat area and isolation."

Drawing on information from wildlife surveys and local interviews conducted at 196 forest fragments spanning a vast region covering 252.670 km2, Dr Peres worked in partnership with Dr Gustavo Canale of the State University of Mato Grosso (UNEMAT). They investigated the effects of anthropogenic landscape alteration and other impacts, such as hunting, on the survival of large vertebrate species.

The researchers travelled more than 205,000km by treacherous dirt roads to uncover the largest and least disturbed forest fragments left in this vast region of the Atlantic Forest.

"We uncovered a staggering process of local extinctions of mid-sized and large mammals," said Dr Canale.

Around 90 per cent of the original Atlantic Forest cover (about 1.5 million km2) has been converted to agriculture, pasture and urban areas, and most of the remaining forest patches are smaller than a football pitch. On average, forest patches retained only four of 18 mammal species surveyed.

This study - the first to document the loss of five large tropical forest mammals from one of the world's most endangered tropical biodiversity hotspots - highlights the critical importance of the few legally protected areas established in the Atlantic Forest.

"We found that the protected areas retained the most species-rich forest fragments in the region," said Dr Canale. "We therefore recommend the implementation of new strictly protected areas, such as National Parks and Biological Reserves, including forest fragments containing populations of endangered, rare and endemic species, particularly those facing imminent extinctions."

However, many of the existing protected areas are far from secure.

Prof Peres said: "A growing number of reserves are being degraded, downsized, if not entirely degazetted, so holding on to the last remaining large tracts of primary forests will be a crucial part of the conservation mission this century."

With the global population projected to surpass nine billion by 2050, tropical forests will face increasing threats posed by anthropogenic land-use change and overexploitation.

"Human populations are exploding and very few areas remain untouched by the expanding cornucopia of human impacts," said Prof Peres. "It is therefore essential to enforce protection in areas that are nominally protected 'on paper'. The future of tropical forest wildlife depends on it."

'Pervasive defaunation of forest remnants in a tropical biodiversity hotspot' by Gustavo R. Canale, Carlos A. Peres, Carlos E. Guidorizzi, Cassiano A. Gatto and Maria Cecília Kierulff will be published online by PLoS One on Tuesday August 14.

Contact: UEA Press Office
press@uea.ac.uk
44-016-035-93496
University of East Anglia

 

 

Marine research in the Brazilian rainforest

Slash and burn practice for centuries as source of stable carbon compounds in the oceans

		This photo shows burning of the rainforest in the Atlantic National Park of Brazil. Credit: Gustavo Luna Peixoto, ICMBio, Rio de Janeiro, Brazil

Until recent decades the Atlantic Rainforest covered a large area of today's Brazil from Amazonas to present-day Argentina. In the 1970s, after years of deforestation, this rain forest was almost completely destroyed, mainly replaced by cattle pastures. This study reveals an unexpected aspect of deforestation. Thorsten Dittmar's team and colleagues from Brazil and the USA show that the common practice of slash and burn left huge amounts of charcoal in the soil. This charcoal is washed out by rainfalls and transported by rivers into the Atlantic Ocean. The soluble fraction of charcoal is composed of extremely stable carbon compounds. The authors conclude that the amounts of these compounds dissolved in the ocean will increase due to human civilization. So far, the effects on marine microorganisms and the global carbon cycle are unknown.

Since way back mankind used fire to shape Earth's vegetation. This was common practice in the 16th century when European settlers came to Brazil, and the beginning of the end of the rain forest. Slash and burn during the centuries reduced its size of 1.3 million to a mere 100,000 square kilometers. What was left was 200-500 millions tons of charred carbon in the soils. These remnants are complex and extremely stable carbon compounds. During the rainy seasons the water elutes the soluble fractions and transports the carbon to the Atlantic Ocean, affecting biogeochemical cycles for centuries and millenia.

In earlier times, the Atlantic rain forest covered a large track of today's Brazil, reaching from 5 to 28 degree south latitude with an area of 1.3 million square kilometers. Until the midst of the 19th century about 95 per cent was intact, but the growing demand for cattle pastures led to massive slash and burn practice which ceased in 1973 leaving only 15 per cent of the original area unaffected. As of today only 8 per cent (100,000 square kilometers) are left.

Thorsten Dittmar and his group at the Max Planck Institute for Marine Microbiology analysed the samples in their laboratories at the University of Oldenburg which are an outpost station of the Bremen based Max Planck Institute. Their main research topic is the carbon cycle and the fate of the dissolved organic matter (DOM) in aquatic, mainly marine, systems. For the sample analysis of the present study they also used their ultrahigh resolution mass spectrometer.

Dittmar explains how they started their rain forest project: "A couple of years ago we detected telltale carbon compounds typical for burnt plant material and charcoal dissolved in ocean water. We speculated that the pre-harvest burning of sugar cane plantations and forest fires in Brazil was a major source. So, we as marine scientists, contacted our colleagues in Brazil, who had taken soil and water samples for years around the Paraiba do Sul river."

		It is the spirit of the destroyed rainforest which you can find in the rivers. Slash and burn: in the tropical forests big trunks are removed, the rest is burnt and huge amounts of charred coal is left in the soil. In 1843 the artist Félix Émile Taunay drew attention to the destruction of the rain forest with his oil painting "View of a native forest that is being reduced to charcoal". (Oil on canvas, 143 x 195 cm). Credit: Museu Nacional de Belas Artes/IBRAM/MinC. (registro n°832) Foto: Jaime Acioli.

The results were surprising. Substantially more carbon compounds where discharged by the river than the annual procedure of pre-harvest burning of sugar cane plantations could produce. "When we plotted the results from the samples against time and compared this pattern with the amount of precipitation and the occurrence of fires, the relation became obvious. The charred carbon must have originated from the slash and burn period from much earlier times."

This assumption was confirmed by further experiments and data. The large scale slash and burn practice ended in 1973. The still practiced pre-harvest burning of the sugar cane plantations produces only 190-740 tons charred carbon annually, but the amount found in the river was three to sixteen times higher. The scientists extrapolated 50,000 to 70,000 tons for the annual load carried by rivers from the total area of the relict rain forest into the Atlantic Ocean. In the laboratory the soil of the rain forest gave the highest yield of carbon compounds in leaching experiments. More and more it became obvious that the sugar cane plantations were not the source, as also high concentrations of charred carbons were detected upstream of the river where only limited numbers of plantation were present.

"There are current discussions in the scientific community about using charred carbon to sequester carbon and thereby removing it from the active global carbon cycle. Our data clearly show that this is not a sustainable procedure, as sooner or later this carbon ends up in the oceans and alters the ecosystem. And we do not know anything about the consequences", Dittmar sums up his concerns. "The soluble fraction of charred carbon is extremely stable in the environment and withstands microbial activities. Therefore, we can trace it in the world's oceans, even in remote places of the deep sea. Our study suggests that this stable fraction of charred carbon in the deep sea will increase due to the increase of anthropogenic activities. The consequences on the marine microorganisms and the global carbon cycle are unknown."

Original work: Thorsten Dittmar, Carlos Eduardo de Rezende, Marcus Manecki, Jutta Niggemann, Alvaro Ramon Coelho Ovalle, Aron Stubbins and Marcelo Correa Bernardes
Continuous flux of dissolved black carbon from a vanished tropical forest biome
Nature Geoscience, 2012. Advanced Online Publication. DOI: 10.1038/ngeo1541

Contact: Dr. Thorsten Dittmar
tdittmar@mpi-bremen.de
49-441-798-3602
Max-Planck-Gesellschaft

 

 

Ship-borne measurements show EU policies have improved air quality in harbours

The European Union

Sulphur dioxide emissions from shipping have sharply decreased in EU ports thanks to an EU policy which limits sulphur content in fuels for ships at berth or at anchor in ports. Scientists at the European Commission's Joint Research Centre measured key air quality parameters in Mediterranean harbours before and after the entry into force of the low-sulphur requirements in January 2010. In European harbours they found an average decrease of 66% in concentrations of sulphur dioxide, a chemical compound that poses risks to health and the environment. Measurements taken in a non-EU port showed that levels of this noxious substance remained the same.

The air quality measurements were carried out using an automated monitoring station on the cruise ship Costa Pacifica which followed a fixed weekly route in the Western Mediterranean during 2009 and 2010.

The concentrations of sulphur dioxide were found to decrease significantly in three out of the four Mediterranean EU harbours that were investigated: Civitavecchia, Savona and Palma de Mallorca; the daily mean concentrations in all of the harbours decreased on average by 66%. JRC measurements in the harbour of Barcelona were inconclusive because of large day-to-day concentration variations. However, independent measurements from monitoring stations in the harbour of Barcelona and in the vicinity of the harbour of Palma de Mallorca confirm a strong decrease in sulphur dioxide concentrations from 2009 to 2010.

In contrast, no decrease in sulphur dioxide was observed in the Mediterranean harbour of Tunis, and there was no reduction in any of the other air pollutants that were measured in all four harbours (Civitavecchia, Savona, Palma de Mallorca and Tunis). This shows that the decreases in sulphur dioxide are a direct consequence of the application of the EU requirements. The study also confirms a correlation between sulphur dioxide and chemical elements typically emitted from ship stacks which demonstrates that ships were the main source of sulphur dioxide in the harbours.

The logistics for the automated ship-borne monitoring station for air pollutants used in this work were provided by Costa Crociere and the measurements were performed in collaboration with scientists from the Universities of Genoa and Florence, the INFN-LABEC Laboratory in Florence as well as from the Institute of Environmental Assessment and Water Research in Barcelona.

Sulphur dioxide is one of the main chemicals responsible for formation of acid rain and particulate air pollution - a major risk factor for cardiovascular and respiratory diseases. The 2005 amendment of Directive 1999/32/EC required that, as of January 2010, all ships at berth or at anchor in European harbours use fuels with a sulphur content of less than 0.1% by weight, while previously, outside of Sulphur Emission Control Areas, a sulphur content of up to 4.5% was allowed. Ships traditionally use heavy fuel oil which, from 2012, can have a sulphur content of up to 3.5% for cargo vessels (before 2012 this limit was 4.5%). The average sulphur content of heavy fuel oil is about 2.4%. By comparison, the sulphur content of fuels used in road vehicles must not exceed 0.001%.

In line with the broader environmental protection objectives of the EU and strengthening a parallel agreement reached by the International Maritime Organisation, the European Parliament and the Council have come to an agreement to be submitted to formal vote after the summer on an amendment to the 1999/32/EC directive to further reduce sulphur content of fuels used outside of harbours. The maximum allowed sulphur content of ship fuels will go down from 3.5% to 0.5% in 2020, and in the Sulphur Emission Control Areas (the Baltic Sea, the North Sea and the English Channel) the current limit of 1,5% sulphur content for ship fuels will be reduced to 0.1% in 2015.

The results of the study, entitled 'Impact of a European directive on ship emissions on air quality in Mediterranean harbours', have been published in the scientific journal Atmospheric Environment. C. Schembari, F. Cavalli, E. Cuccia, J. Hjorth, G. Calzolai., N. Pérez, J. Pey, P. Prati, F. Raes: Impact of a European directive on ship emissions on air quality in Mediterranean harbours, Atmospheric Environment (2012), doi: 10.1016/j.atmosenv.2012.06.047

Contact: Berta Duane
berta.duane@ec.europa.eu
39-033-278-9743
European Commission Joint Research Centre

 

 

Mysterious snake disease decoded

UCSF team uncovers possible cause of deadly illness -- a virus that was completely unexpected

		This is Balthazar the snake. Credit: Jason Socrates Bardi/UCSF

A novel virus has been identified as the possible cause of a common but mysterious disease that kills a significant number of pet snakes all over the world, thanks to research led by scientists at the University of California, San Francisco (UCSF)—and three snakes named Juliet, Balthazar and Larry.

The virus, previously not thought to infect snakes at all, appears to cause "inclusion body disease." Long the bane of zoo officials and exotic pet owners, the deadly illness spreads among boas and pythons in captivity, causing micro clumps of clustered proteins to form inside the snake, leading to bacterial infections, neurological problems, anorexia and withering, leading to death.

The new work, described this week in the American Society for Microbiology's new open-access journal mBio, paves the way toward developing diagnostics and treatments, which may make it possible to eradicate the disease from snake collections worldwide.

"It's a devastating disease when it gets into a collection, zoo or aquarium because it's essentially fatal every time," said Joe DeRisi, PhD, the senior author of the study, a Howard Hughes Medical Institute investigator (HHMI) and vice chair of the Department of Biochemistry and Biophysics at UCSF.

Surprisingly, he said, the cause of the illness appears to be a completely new set of viruses of a type known as an arenavirus. The discovery came as a complete a shock to the team of scientists because, while arenaviruses are common in rodents and cause extremely nasty infections in other mammals, nobody knew they could infect reptiles.

"Now we have found that they infect snakes, as well," said Mark Stenglein, PhD, a postdoctoral fellow at UCSF who is the first author on the paper.

Stenglein, DeRisi and their colleagues isolated at least two strains of the arenaviruses from half a dozen snakes afflicted with inclusion body disease. They could find no traces of the same viruses in snakes that were free from disease.

Arenaviruses infect mostly rodents but occasionally people, and can cause fatal hemorrhagic diseases like Lassa fever, which kills thousands of people every year in Africa. There is no evidence, however, that a snake has ever transmitted an arenavirus infection to a person despite the fact that snake owners and veterinarians handle infected snakes all the time, said DeRisi.

Three Snakes That Shook the World

For years, many experts have hypothesized that a virus or some other infectious pathogen might cause inclusion body disease because of evidence that it spreads easily from snake to snake. No definitive cause has been identified until now, and the discovery may never have occurred if not for a random sequence of events, including cases of inclusion body disease in an aquarium collection, a friendly DNA sequencing competition among scientists, a postdoctoral researcher who was looking for a project, and a snake owner worried about her favorite pet.

The story began with a snake named Larry, and his owner, Taryn Hook of San Jose, California. Before Larry, Hook had lost two snakes to inclusion body disease, and, in early 2009, she became convinced Larry had it as well. He was developing bacterial infections similar to what Hook had seen with her two other snakes. Knowing there was no treatment or cure, she was desperate to find anyone who might save her snake.

Hook took Larry to see the exotic pet veterinarian Chris Sanders, DVM, owner of the nearby Wildwood Veterinary Hospital. Sanders had just attended a conference at which he had heard DeRisi talking about his Virochip DNA microarray technology and its ability to identify viruses, fungi and other pathogens—including at least one exotic pet disease, a mysterious parrot virus—when no other gene probing technology could. The parrot virus was discovered by DeRisi and Don Ganem, MD, at the time an HHMI investigator and professor of microbiology and medicine at UCSF, in 2008.

Could DeRisi help save Larry the snake as well? Sanders suggested to Hook she had nothing to lose by asking.

DeRisi was in his office one morning in early 2009 when he spied a hand-written letter in his stack of daily mail. Inside was a plea from Hook describing Larry's illness. She had heard he had found the cause of a mysterious parrot disease. Would he do for snakes what he did for parrots? She enclosed a picture of herself with Larry.

Having never heard of the disease, DeRisi set the letter aside and it was lost under a pile of paper. Only months later, while cleaning his office, did he stumbled across it again. He was about to toss it away, but in scanning the letter again he noticed Hook mentioned the local exotic pet veterinarian Sanders. So he called, they spoke, and DeRisi decided to take on the project.

"It satisfied all the criteria as an interesting disease," DeRisi said. But first he had to find samples to test from infected snakes.

		This is Joe DeRisi, Ph.D. Credit: UCSF

Around the same time, inclusion body disease was diagnosed in a snake at the Steinhart Aquarium in the California Academy of Sciences in San Francisco. Also discovered were snake mites, which are believed to be a possible vector for passing the disease from snake to snake.

Academy veterinarian Freeland Dunker decided to test all of the boas exposed to the infected snake for the disease—a complicated procedure requiring a surgical biopsy of the liver. He discovered a few more were infected, and all of them had to be euthanized to prevent any spread of the disease. Dunker asked his pathologist, Drury Reavill DVM if she knew of any current research being done on inclusion body disease for which tissues from the euthanized snakes could be used. As it turned out, Reavill had already been in touch with DeRisi's group and knew they were looking for samples.

The effort to find the virus went into overdrive after Stenglein joined the DeRisi laboratory as a postdoctoral fellow and took on the project. But before he and DeRisi could find traces of the virus they needed to know the sequence of the boa constrictor genome so they could distinguish snake DNA sequences from viral sequences in the diseased animals. The problem was that there were no snake genomes available.

Thus, their first step was to sequence the entire boa constrictor genome, and they had to start with a snake that they were sure was free of inclusion body disease. At the Academy, Dunker helped in this effort by collecting blood from a boa constrictor named Balthazar, an education animal which was housed separately, had no contact with the rest of the boa snake collection and tested negative for the inclusion body disease.

Substantial help in the sequencing effort came from scientists participating in a friendly competition called the Assemblathon 2, which was sponsored by UC Santa Cruz and UC Davis. Balthazar's DNA was sequenced and a number of groups around the country competed to build (assemble) the most complete genome sequence possible using the raw data.

Characterizing Balthazar's genome paved the way for finding the arenavirus in the midst of millions of other sequences of the snakes' DNA. This "needle in the haystack" problem was solved using custom software written in the DeRisi lab, and made available for free on his website.

This is where the shock came.

The team found two arenavirus strains in the snakes—a surprise in itself; but in addition, they observed that theviruses did not look like your ordinary arenaviruses. They looked like distant relatives of other arenaviruses but had protein coats that were more similar to those of Ebola viruses. Like arenaviruses, Ebola virus can cause fatal hemorrhagic fever when transmitted to humans. Neither of those viruses has ever been known to infect reptiles, and although it had been postulated that they share a common ancestor, no such virus linking them had ever been discovered.

Once the virus was computationally identified, the team had to find a way to grow the virus so that it could be studied further. Because the virus infects boa constrictors, the ideal way to grow it, the team reasoned, would be to infect boa constrictor cells, but no such cell line existed. So DeRisi and Stenglein turned to a third snake, named Juliet.

Juliet was a red tailed boa owned by Chris Sanders, who'd had her since his days as a young veterinary student. She was about 20 years old when Balthazar's genome was being assembled and was dying of lymphoma. When she ultimately succumbed to the cancer, Sanders harvested her kidneys and the DeRisi laboratory was able to use them to make a boa constrictor cell line.

The scientists took virus from diseased snakes, added it to Juliet's kidney cells growing in petri dishes and showed that the snakes accumulated exactly the same "clumps" of proteins as had been observed in the sick snakes from the Academy. Antibodies raised against the virus showed that these clumps were formed from arenavirus protein, further strengthening the association of this new virus and the deadly disease.

In solving this longstanding veterinary mystery and setting the stage for treatments, vaccines, and perhaps even eradication of this disease, the scientists also discovered an unexpected new branch of virus biology: The viruses they found appear to be a combination of arenaviruses and filoviruses, neither of which has been known to infect reptiles.

The article, "Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: a candidate etiological agent for snake inclusion body disease (IBD)" by Mark D. Stenglein, Chris Sanders, Amy L. Kistler, J. Graham Ruby, Jessica Y. Franco, Drury R. Reavill, Freeland Dunker, and Joseph L. DeRisi is published in the journal mBio on August 14, 2012.

In addition to the authors from UCSF, authors of this study are affiliated with Wildwood Veterinary Hospitals in Portola Valley, CA; Novartis Institutes for Biomedical Research, Zoo/Exotic Pathology Services in Sacramento, CA; and the California Academy of Sciences in San Francisco. This work was supported by the Howard Hughes Medical Institute and the National Institutes of Health through grant # 5T32HL007185-34, and PSWRCE, NIH grant U54 AI065359.

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Contact: Jennifer O'Brien
jennifer.obrien@ucsf.edu
415-502-6397
University of California - San Francisco