THE USE OF A VIABILITY ASSAY TO FORMULATE A
NEW
COCCIDIOSIS VACCINE
Julia J. Dibner, Marco A. Quiroz, and Chris D. Knight
Novus International, Inc., 20 Research Park Dr., St. Charles, MO 63304
marco.quiroz@novusint.com
Determination of coccidial oocyst viability has
traditionally been done using in vivo methods such as lesion score or oocyst
shedding following the oral delivery of a dose determined by microscopic
enumeration. The microscope is readily used to determine number of sporulated vs
non-sporulated oocysts per ml, but the determination of % live oocysts is not
possible using a microscope. This introduces a source of uncontrolled
variability when multiple suspensions of sporulated oocysts are used to
formulate a live coccidiosis vaccine. An in vitro assay (VIACYST® assay) has
been developed to measure the viability of sporocysts of E. tenella, E. maxima
and E. acervulina. The assay is based on the use of a non-vital stain, ethidium
bromide. The assay requires the excystation of sporocysts and depends on the
permeability of sporocysts from Eimeria or any other sporocyst-forming protozoan
to this non-vital dye. Preliminary experiments confirmed that vaccine from a
suspension of E. tenella oocysts assessed non-vital by ethidium bromide staining
were also not capable of generating resistance to a coccidiosis challenge. As
the proportion of oocysts from the non-vital suspension increased in the
vaccinating dose, there was a concomitant decrease in the resistance of the bird
to oral challenge as indicated by lesion scores. When the immunizing inoculum
contained 1000 viable oocysts or less, lesion scores were not different than
non-vaccinated controls. This study confirmed that the in vitro viability assay
correlates to the ability to confer resistance to a coccidiosis challenge. A
second experiment was done to test the use of the viability assay to formulate
vaccines from suspensions of oocysts that ranged from 24 to 52 weeks old and 16
to 82% viability. Results indicated that suspensions of E. acervulina varying
widely in viability and age were all capable of generating resistance to an oral
challenge providing the vaccine was formulated on a viable oocyst basis.
Ethidium bromide assay detects non-viable sporocysts that reflect the original
oocyst population and that formulation of the vaccine based on viable sporulated
oocysts/bird will yield consistent efficacy across a wide range of age and
viability.
®VIACYST is a service mark of Viridus Animal Health, LLC, and is registered in the United States and other countries.