In vitro development of cyathostomin larvae from the third stage larvae to the fourth stage: morphologic characterization, effects of refrigeration, and species-specific patterns

A mixed population of equine cyathostomin (Nematoda, Strongyloidea) infective third stage
larvae (L3) was cultured in vitro using a cell-free medium. Some L3 were cultured
immediately after Baermann collection fromfecal cultures, while others were kept in water
at 4 8C for 7 days before initiating the in vitro cultures. Cultures were examined daily for
viability. At days 2, 7, 14 and 21 larvae were collected for identification of developmental
stage and morphological changes, using both light and scanning electron microscopy. Larvae
were classifiedas early L3(EL3), developing L3(DL3), late L3(LL3)andfourth stagelarvae(L4)
on the basis of morphological features. Viability remained high throughout the entire study
period in cultures of both non-refrigerated (84.7%) and refrigerated (77.4%) larvae.However,
viability of the non-refrigerated was significantly greater from7 through 21 days of culture.
Significant differences were also observed in the percentage of DL3 between the nonrefrigerated
and refrigerated larval cultures by day 7. The highest percentage of DL3 larvae
(22.5%) was reached at the end of study in those larvae that were not previously refrigerated.
The data suggests that prior refrigeration decreases viability and slows L3 development. At
day21 LL3 larvae were only asmall percentage of theDL3: 6.9 and 5%innon-refrigerated and
refrigerated cultures, respectively. Few of these larvae freed themselves fromthe L3 cuticle
andmoulted to L4 stage. Characteristics of individual species in vitro developmental patterns
were determined by the molecular identification of individual larvae in pools of larvae
randomly collected at days 0 and 21. Seven species (Coronocyclus coronatus, Cylicostephanus
goldi, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus
ashworthi, Petrovinema poculatum) were identified in the day 0 pool. The greatest
tendency to develop in vitro was shownby the genus Cylicostephanus with the species C. goldi
and C. longibursatus that developed to the LL3-L4 stages. C. nassatus, C. ashworthi and C.
coronatus did not progress in their development beyond the EL3 stage, while no apparent
signs of development were registered for C. catinatum.