Growth of Philophthalmus sp. (Trematoda) in the Eyes of Chicks

Abstract

The in vivo growth pattern of Philophthalmus sp., an ocular trematode, was determined by median slit or intraocular inoculations of one to ten larvae into 65 chicks. None to three flukes per eye were recovered, relaxed in mentholated alcohol, and measured live at almost daily intervals from 2 to 65 days after exposure. The rate of growth, slow until the 5th day (average length from 0.76 to 1.00 mm at 5 days), became rapid (reaching 2.75 to 3.25 mm) until about the 20th day, and when fertilization occurred, continued at a slower rate (4.00 to 4.50 mm at 55 days). Monometacercarial infections suggest that selffertilization does not occur. Flukes not fertilized fail to grow beyond the 20-day level. Larvae inoculated into one eye sometimes migrate into the other eye. Developmental stages based on well-defined morphological characteristics were assigned to uniformly fixed, stained, and mounted flukes. The five in vivo stages were as follows: metacercarial or undifferentiated stage from 0 to 4 days; gonadal differentiation stage from 4 to 10 days; preovigerous stage from 10 to 13 days; ovigerous stage from 13 to 65 days, and embryonated egg stage from 21 to 55 days. Only fertile worms that contained miracidia with eye spots were included in the fifth stage. Brief mention has been made earlier of a marine species of Philophthalmus which reaches sexual maturity under the nictitating membrane of a variety of avian hosts (Penner and Fried, 1961). The growth pattern of this species in the eyes of chicks has been determined and reported in abstract (Fried and Penner, 1961). This paper presents details of the in vivo study. MATERIALS AND METHODS Batillaria minima (Gmelin) snails from Florida naturally infected with Philophthalmus sp. were obtained from Dr. L. R. Penner. Snails were maintained in aquaria (Wagner, 1960) or kept dry until ready to use and then placed in finger bowls half-filled with sea water for cercarial emergence. Encysted metacercariae or those excysted with a scalpel were used up to 3 weeks after encystment. Connecticut Randombred (King et al., 1959) or White Leghorn chicks were obtained from the Poultry Science Department and used from day of hatching to 7 days old. Fifty-four chicks were exposed to one, two, five, or ten excysted metacercariae via the median slit (oral opening of naso-lachrymal ducts) as follows: a chick was turned on its dorsal side, its head deflected at a Received for publication 30 October 1961. *From a dissertation submitted in partial fulfillment for the requirements of the Ph.D. degree at the University of Connecticut. This work was supported in part by U. S. Public Health Service Grant E-740 to the University of Connecticut. fPresent address: Department of Biology, Emory University, Atlanta, Ga. 45 degree angle, and an attenuated pipette containing the metacercariae in a minimum of Ringer's solution was placed about 2 mm into the slit. Eleven additional chicks were exposed intraocularly to four to ten metacercariae. The chicks were anesthetized with intramuscular injections of Equi-Thesin (Jensen Salsbery Laboratory, Inc., Kansas City, Mo.). A dosage of 0.025 ml per g of body weight recommended by Gandal (1956) for chickens was effective for chicks. A bird's nictitating membrane was lifted with a watchmaker's forceps and the larvae in a small amount of Ringer's were inoculated under the membrane with a fine pipette. Excess fluid was drained from the corner of the eye, and any larvae washed out were reinoculated. Chicks were kept under usual animal room conditions and examined from 2 to 65 days after exposure, at almost daily intervals up to 35 days, and at approximate 5-day periods beyond that. In performing autopsies, a chick was killed by decapitation and its head mounted on a crossshaped board with two elastics. The board was secured to a dissecting scope also with elastics. The upper and lower eyelids were removed and the nictitating membrane was deflected with a watchmaker's forceps and incised with fine scissors close to the sclera. The membrane was placed in a petri dish containing Ringer's, and flukes were dislodged from the membrane's interior surface with needles and transferred to fresh Ringer's. Eyes were treated individually to keep track of their identity. The head was also bisected sagitally and each half placed in a petri dish and examined for eye flukes that might not have been removed by the first technique. Eye flukes were relaxed, measured live, fixed, and stained as follows: a drop of stock mentholated alcohol (240 per cent menthol dissolved in 10 ml 95 per cent ethanol, Abdel-Malek, 1951),