Cover Story - Know the Spore

Identifying viable, sporulated oocysts key to a quality coccidiosis vaccine

Knight: 'It's critical to have the right balance of antigens"�'

Identifying viable, sporulated Eimeria oocysts and ensuring that birds get just the right amount is one of the most important steps that go into producing an effective coccidiosis vaccine.

Even so, with thousands of chicks moving through the hatchery, it's difficult for busy poultry veterinarians and production managers to appreciate the technology, careful selection process, experience and rigid quality-control standards that need to go into a vaccine of this nature.

"Coccidiosis vaccination works by providing a controlled, carefully balanced dose of oocysts "coccidial eggs "to protect against the several species of Eimeria that cause the disease in birds," says Graham Knight, manager of coccidiosis vaccine production at Schering-Plough Animal Health Corporation's production plant in Millsboro, Delaware.

"It sounds easy, but there's more to it than gathering oocysts and putting them in a bottle. The preparation and identification of suitable oocysts is crucial," he says. "It's also critical to have the right balance of antigens, which are the individual components that lead to protection against the Eimeria species causing disease."

Stimulating immunity

Producers need to understand that for a coccidiosis vaccine to stimulate immunity, oocysts must be capable of releasing viable spores. "In other words, they must be sporulated. They also must be viable to be infective," Knight says.

"An infective oocyst is a sporulated oocyst, but a sporulated oocyst is not necessarily an infective one," Knight says. "We think this is an important distinction for producers to know and understand."

The reason is that sporulated oocysts age and die. In addition, some oocysts never fully develop; they are only partially sporulated, while others may be abnormal or damaged and are not infective.

"A major part of our job is the identification of fresh, fully sporulated oocysts," Knight says. "These are the oocysts that are infective and that convey immunity to birds against coccidiosis. It takes a lot of experience to build a quality coccidiosis vaccine."

Tried and true

Although it's a tedious and important process, identifying viable sporulated oocysts isn't rocket science, Knight insists, but it does require an educated eye.

"In Millsboro, we have well-trained, seasoned technicians "many have been involved since the operation was moved to Millsboro in the 1980s. With a good microscope, they can easily differentiate a fresh, fully sporulated oocyst from an oocyst that's not," he says.

Microscopic examination is the traditional method of determining whether an oocyst is fully sporulated and has been in use since the early 1900s. "It's a straightforward and proven method, one that requires no manipulation of the sample other than dilution and needs no specialized equipment or technique," Knight explains.

Dr. Steve Fitz-Coy, now a technicalservice representative for Schering- Plough Animal Health, agrees.

"Poultry producers have been dealing with coccidiosis for nearly 75 years, since groundbreaking work by E. E. Tyzzer in 1929. Identifying sporulated oocysts with a microscope is a tried and true process that's improved with time," he says.

Knight points out that data collected over the course of many years as well as field experience have demonstrated that coccidiosis vaccine made from fresh, fully sporulated oocysts within a fixed time frame has enough viable oocysts of each species at the end of the stated shelf-life (12 months) to be efficacious.

Complementary procedures

Several other procedures built into coccidiosis- vaccine production at Millsboro complement the identification of viable, sporulated oocysts and contribute to the vaccine's efficacy, says Knight. One is the way in which oocysts are produced. Each coccidial species in the vaccine is grown in birds in a room dedicated to that species. The rooms are located in an antigen-production facility. The birds are not used for multiple species nor are they re-used, he says.

"Although most species of coccidial oocysts can be differentiated by trained technicians, there are exceptions. Different species have different sizes and shapes, but some overlap occurs, particularly among small oocysts. Growing each type in isolation solves this problem," Knight says.

Technicians inoculate one group of birds with known and tested seeds of one coccidial species. Excreted oocysts are then harvested and cleaned. "We don't need to differentiate oocysts - we only have to determine whether an oocyst is fully sporulated or not and how many there are.

"We need different numbers of oocysts for each species to make Coccivac. Ultimately, the oocysts are blended, but we grow them individually," he says.

Oocyst production

Nevertheless, Fitz-Coy points out, oocysts excreted by birds are not sporulated when harvested and, therefore, are not infective.

"We have to transform the harvested, non-sporulated oocysts to the sporulated and infective form under controlled conditions by providing warmth, humidity and oxygen," he says.

Adds Knight, "We mimic nature, but with the benefit of controlled conditions, we can efficiently transform the majority of oocysts over a given period of time. Because the viability of sporulated oocysts decreases with time, the harvested material is processed quickly at the Millsboro plant, which works to cGMP (current Good Manufacturing Practices)."

After sporulation, the antigen lot is sampled and technicians identify and count the number of infective oocysts per ml. "We get at least two independent counts on freshly produced material using light microscopy," Knight says.

The final product includes nonsporulated and partially sporulated oocysts, but only oocysts that are fully sporulated are counted as infective, he says.

Fitz-Coy says, "We must keep in mind that oocyst viability decreases over time. Not all originally counted infective oocysts will be viable by the expiration date on the vaccine."

Consequently, the Millsboro team makes sure that every vaccine formulation contains sufficient infective oocysts to allow for the decay of some infective live oocysts. "That way, the vaccine maintains potency through its stated shelf-life," he says.

Antigen production is complete once the harvest has been treated with a chemical sterilant and quality control has released the lot on sterility, purity and titer, he says.

Potency testing

To be absolutely certain every final vaccine serial (a blend of individual vaccine lots) is efficacious, technicians conduct potency testing in live birds, says Knight. "Vaccinated birds are challenged with each Eimeria line to make sure immunity has developed. Unvaccinated control birds also are challenged to ensure the validity of the test."

Besides making sure that every batch of vaccine contains enough viable, sporulated oocysts, the count also ensures that birds aren't exposed to too many, which could cause a stronger than needed immune response.

"Actually, the formulation ensures that fresh vaccine is not too potent," says Knight, "but as an extra precaution, we conduct safety tests in birds at an increased dose level.

"The nature of the potency test makes it both time- and resource-consuming. It also eats into the available expiration dating of the product. However, we still believe that a challenge potency test is the best way to demonstrate vaccine efficacy," he says.

Additional testing

Every batch of Coccivac also is tested for the presence of extraneous viable bacteria and fungi, according to USDA regulations and, for added measure, for mycoplasma even though this testing is not a government requirement. "Although the potency test is critical because it demonstrates that all species are present in sufficient numbers to initiate the immunizing process, that alone is not enough to warrant release of the vaccine," he says. Further testing is carried out to demonstrate freedom from contaminating viral agents.

The effectiveness of Coccivac is ensured by a range of measures starting with the quality of raw materials and ending with shipment of the product, according to Knight.

"Every single stage of production, be it a quality-control test, an inprocess check, an incubation temperature or storage time, is documented and reviewed for compliance prior to

Source: CocciForum Issue No.7, Schering-Plough Animal Health.

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