Cover Story - 'This is the Future'

IAH's Martin Shirley is leading the way to mapping the Eimeria genome and developing an even better understanding of coccidiosis in poultry

Shirley and Tomley: 'By sequencing Eimeria DNA, or unraveling its genetic code, we can open up the parasite"�and come up with thousands of potential targets for control.'

For the moment, coccidiosis can wait.

Dr. Martin Shirley has just put in another long day at his lab in Compton, England, where the world-renowned coccidiologist serves as principal scientist for the Institute of Animal Health's division of molecular biology.

After stopping home to squeeze in some house painting and taking a long walk through the quiet countryside, he's ready to unwind at a pub in his hometown of Wantage "a quaint 17th century village some 50 miles northwest of London.

Sipping on a frothy pint of his favorite beer, he enthusiastically discusses his latest accomplishment: Converting more than 7,500 songs from his personal CD collection to the MP3 format and cataloging them by artist and genre on his home PC, which is wired to a state-of-the-art sound system.

"And I just found a new program that will allow me to transfer all the old stuff I have on vinyl to MP3 while eliminating all the pops and scratches," he says, clearly embracing the new technology. "So when I'm all done, I'll have everything from Dylan, Sam & Dave and the Stones to Beethoven, Mozart and Verdi completely catalogued. Every song will be only a mouse click away."

Cataloging coccidiosis

The following day, when he returns to his lab at IAH, Shirley is all business. He's still working at his PC, only this time he's labeling and cataloging the DNA of Eimeria parasites, not the vintage recordings of Led Zeppelin or Stevie Wonder.

"This is the future of coccidiosis management in poultry," he says confidently.

"In human medicine, if you look at the number of drugs available for controlling diseases, we can effectively hit about 470 biological targets. That sounds like a lot, but not when you consider that our bodies produce something like 30,000 to 40,000 gene products. We still have a long way to go. That's one of the attractions of the human genome project. Scientists are opening up the whole genome, so potentially, we can find and then tackle every gene that's linked to a particular disease situation.

"The same is true for Eimeria in poultry," he adds. "By sequencing Eimeria DNA, or unraveling its genetic code, we can open up the parasite for public display, look at it, dissect it and come up with maybe thousands of potential targets for control in the future."

'Complicated' organism

Shirley notes that Eimeria parasites are "complicated" organisms with perhaps up to 10,000 gene products or, put another way, 10,000 targets for either direct chemotherapeutic or biological control.

"At the moment, we have a small portfolio of coccidiosis drugs and vaccines that, while very effective, probably target no more than a half dozen biological targets, perhaps 10 at the most.

Coccidiosis Control: How Good Can It Get?

It could be argued that the poultry industry is already achieving good or at least adequate control of coccidiosis with current drugs and vaccines. Still, Shirley sees plenty of room for improvement.

"There's a need for products to replace those that are becoming less effective, especially the older drugs that have been showing resistance," he says. "We also have politics to consider. Regulators in Europe and other parts of the world now have a profound interest in the drugs used to control diseases of poultry and livestock, and some products for coccidiosis have been withdrawn. So we're left with a small portfolio of drugs and vaccines for a disease that's ubiquitous in poultry flocks throughout the world."

Shirley says that poultry flocks are generally infected with large numbers of coccidial parasites.

"In the UK, with drug programs, we're often talking about 60,000 parasites per gram of litter. That's every gram of litter," he says. "A poultry house might contain 2 to 3 tons of litter, so that's a huge number of parasites developing in the face of control. Moreover, since parasites have enormous replication rates there is potential for a lot of mutation. The process may not be as fast as it is with viruses, but Eimeria have an affinity to change profoundly."

Shirley also considers that the poultry industry needs to be careful when setting parameters for "good" coccidiosis control, especially with vaccines that operate quite differently from drugs.

Shirley thinks vaccines, not drugs, will pave the way to new breakthroughs in coccidiosis management. "We're already seeing that with the development of Paracox™ and the tremendous growth of other coccidiosis vaccines like Coccivac," he says. "Developing vaccines against parasites is a sophisticated process and presents a very difficult challenge."

"That's one of the reasons we're sequencing the DNA of Eimeria," he continues. "For all the progress we've made against coccidiosis in poultry, we really know nothing about the finer points of the biology of Eimeria parasites. For example, almost nothing is known about metabolic pathways, the mechanisms by which the parasite damages the host or of the molecules that stimulate protective immunity (i.e., how and why vaccines such as Coccivac and Paracox are so effective). All of this information is contained in the genome sequence."

Shirley says his research team at IAH is concerned primarily with molecular aspects of the Eimeria genome, as well as the genetics of the parasites.

"Without doubt, the most exciting spin-off from our work has been our recent success in securing the funding of a genome sequencing project for Eimeria tenella, which causes cecal coccidiosis in chickens," he says. "This is a fantastic outcome for the coccidiosis community worldwide, as E. tenella has become the first protozoan of global veterinary importance to be sequenced on a large scale."

Dream team

In March 2002, Shirley, IAH colleague Dr. Fiona Tomley and Drs. Bart Barrell and Al Ivens from the Sanger Institute, Cambridge, were awarded a grant of £750,000 (US$1.2 million) from the UK's Biological and Biotechnological Science Research Council to determine the DNA sequence for the world reference Houghton strain of E. tenella.

"Our previous work has shown that the genome of E. tenella comprises about 60 million base pairs of DNA contained within 14 chromosomes," says Shirley, who expects to wrap up the project by June 2004. "This amount of DNA may give rise to around 8,000 to 10,000 different proteins. But at present, literally only a small handful of these proteins have been identified and only very few of the genes responsible have been characterized."

When the project is finished in June 2004, Shirley's team will have assembled a genetic blueprint for E. tenella and revealed 90% of the parasite's encoded proteins. All the data generated by the project is being posted on the Internet and available to the public. Shirley says the data will allow current and future coccidiologists to identify new targets for vaccination and chemotherapy.

Eimeria's 'lifestyle'

"The data will yield a much greater understanding of how Eimeria parasites go about their lifestyle "for example, how they cause disease, find the correct parts of the gut in which to develop, get into the host cells, reproduce themselves, cause the host to develop immune responses, and a myriad of other biological features," Shirley explains.

"In addition, the data will allow the biology of Eimeria parasites to be compared with that of close relatives, such as Plasmodium (the malarial parasites), Cryptosporidium, Neospora and Toxoplasma. If one of these parasites invades in a particular way, for example, you can be sure that Eimeria probably invades the host cell in a similar way. In the research community, there's a lot of mixing and matching between these different organisms."

The sequencing initiative under way at the IAH and the Sanger Institute was provided with many letters of support from international scientists and veterinarians working on coccidial parasites and coccidiosis. Shirley says the funding of the work by the BBSRC represents a major push for veterinary science. "To date, the genomes of very few pathogens of purely veterinary importance have been sequenced, and none remotely as big as E. tenella" he says.

But Shirley and his colleagues at IAH and Sanger are not going at this alone. The UK scientists are also collaborating with sequencing efforts by Dr. Arthur Gruber in São Paolo, Brazil, and Dr. Wan Kiew Lian at the Universiti Kebangsaan, Malaysia.

"Arthur is doing some great research that dovetails in to the big sequencing initiative and Wan has been awarded a grant from the Malaysian government to derive the complete sequence of chromosomes 1 and 2 (each of 1 million base pairs of DNA)," Shirley says.

"These chromosomes will be sequenced in their entirety to capture all genes and, most interestingly, chromosome 2 is linked to the trait of precocious development that characterizes the attenuated parasites used in Schering-Plough Animal Health's Paracox, the current attenuated vaccine."

Why Target Eimeria?

Eimeria is the most economically significant family of parasites for poultry and intensively reared livestock, according to Dr. Martin Shirley of IAH.

"Coccidiosis has most impact in the intensive poultry industry, where all 35 billion chickens raised annually are likely to become infected," he says. "Despite routine prophylaxis with anticoccidial drugs, coccidiosis costs the UK poultry industry alone around £40 million ($64 million) a year, which is equivalent to 4.5% of the revenue from sales of live broilers."

Subclinical coccidiosis is commonplace because the efficacy of drugs is severely compromised by drug-resistant parasites, Shirley says. In the EU, coccidiosis is also considered a "severe welfare problem" causing malabsorption, weight loss, diarrhea, hemorrhaging, anemia and death.

"Eimeria tenella is one of the most common and pathogenic species that infect the domestic chicken and the disease of cecal coccidiosis is one of the most highly visible aspects of coccidiosis in poultry," he says. "Coccidiosis control is complex and it seems clear that sustainable control will rely increasingly on vaccination, either alone or in combination with drugs."

But don't expect coccidiosis to be eradicated any time soon.

"Eimeria is fantastically suited for survival and replication, especially in circumstances "warmth, moisture, high bird density "provided so wonderfully by the poultry industry," Shirley says. "For example, the oocysts have a transmission stage that is extremely robust and tough. In fact, in the laboratory, we can incubate these in bleach and the parasites still survive very well."

"The parasite itself is a bit like a Russian doll, with four discrete genomes inside it," he adds. "Once the Eimeria parasite gets into the host, it really kicks in with a large range of genomes."

Solving the Eimeria Puzzle

Scientists use one of four letters "A, C, G or T "to identify each DNA molecule within a chromosome. These letters occur in pairs (A with T and C with G) and, in total, the 14 chromosomes in the Eimeria organism contain about 60 million such pairs of DNA.

"This genomics project will eventually give us a precise sequence of A, C, G or T in Eimeria's 14 chromosomes to create a blueprint of the Eimeria genome," Shirley says.

It's a long and tedious process, however, and one that involves, firstly, breaking each chromosome into many small pieces at random and, secondly, deriving the DNA sequence of each small piece.

"The computer then becomes very important as it examines each individual genome sequence read and works out how the random, overlapping pieces were originally assembled in the chromosome. Eventually the computer compiles a very large map for us," he says. "It's a bit like putting a jigsaw puzzle together."

One of the more tricky parts of the work of the computer is to deal with the characteristic repeats in the E. tenella chromosomes (e.g., GCAGCAGCAGCA).

"You only have to go through several hundred base pairs of sequence before you turn up a repeat," Shirley explains. "The chromosomes of Eimeria are very distinctive in that they contain an abundance of repeats "� but as yet we have no real idea as to the effect of these repeats on the biology of the parasites. Clearly, they must do so as we know that some genes are characterized by an abundance of the characteristic small repeats."

One of the research group's objectives is to see the identification of potentially new targets for vaccines or drugs against coccidiosis. For example, animal health company researchers could scan the Eimeria genome looking for clues for particular proteins or other features compatible with their product-development objectives.

"If they have compounds that they think are active against a particular molecular target, they can screen the E. tenella database to determine if the same target is present in this coccidian," Shirley explains. "Ultimately, it is certain that the genome data will be used to effect better control of the avian coccidia."

Source: CocciForum Issue No.6, Schering-Plough Animal Health.
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