Relationship between the Number of Circulating Microfilariae and the Total Population of Microfilariae in a Host

Abstract

The levels of circulating microfilariae in dogs infected with Dirofilaria immitis remain relatively constant after infusion or removal of large numbers of microfilariae. When uninfected dogs were injected with microfilariae, only 7% were present in the peripheral circulation, if their number was calculated from the animal's blood volume and level of microfilaremia. Removal of a number of microfilariae greater than that calculated to be in the circulation of such a dog resulted in no change in microfilaremia. It is apparent that the regulation of levels of microfilariae is unrelated to the presence of adult worms. Most microfilariae are evidently sequestered in capillaries, or possibly in the tissues in extravascular sites, and have ready access to the circulation. The number of microfilariae (MF) detected in a sample of blood from an infected host does not vary greatly with time, within a range of daily or seasonal variation characteristic of a species. This characteristic stability has also been noted in experimental animals that received MF by transfusion and harbored no adult worms (Hawking, 1967; Hawking et al., 1965). The size of MF-5 to 6 pm in diameter and 100 to 300 /i in length-and their motility suggested that potentially they could be randomly distributed throughout the macroand microcirculatory systems of the body. If so, their distribution might to some extent be governed by simple physical rules relating size of the MF, diameter of vessels, and flow of blood or lymph. Transfusion of MF into an uninfected host has been one of the principal approaches in studies of the relationship between MF and the vertebrate host. It is significant that in the many transfusions that have been made, the proportion of transfused MF detected in blood is usually reported to be between 5 and 20% (Hawking, 1967; Wong, 1964). Studies with Dirofilaria corynodes (Pacheco and Orihel, 1968), D. immitis (Greenough and Buckner, 1969), as well as reevaluation of the results obtained by Wong (1964) and Hawking (1967) indicated that a relatively small proportion of the total population of MF was present in the peripheral circulation at any Received for publication 10 June 1974. * Although submitted in June 1974, this ms was last seen by the author in July 1972. Dr. Pacheco died 24 April 1974 after a long illness. given time. To further test this hypothesis, and simultaneously to get a better estimate of the total number of MF present in a host, (1) complete exchange transfusions were attempted in three dogs and (2) massive numbers of MF were repeatedly injected into two others. MATERIALS AND METHODS Five dogs were used in the experiments: two adults infected in nature with D. immitis, one adult infected in nature with Dipetalonema reconditum, and the last two, born in the laboratory, had not been exposed to vectors of filariae at any time; MF could not be demonstrated in concentrates of their blood prior to the experiments. MF for all injections were isolated and concentrated by Wong's (1964) technique from donor dogs whose level of microfilaremia was more than 1,000 per 20 mm3 of blood. MF to be injected were suspended in 10 to 20 ml of Krebs-Ringer bicarbonate (Umbreit et al., 1946). Except during the exchange transfusions, blood samples used to estimate microfilaremia were taken by puncture of a lateral ear vein on all of the dogs. Free-flowing blood only was used to fill disposable 20-mm3 pipettes. When present in sufficient numbers, MF were counted by a micro-modification of the Knott (1939) technique in which 20 mm3 of blood was lysed on a standard 3by 1-inch microscope slide in 80 mm3 of 2% formalin, mixed thoroughly, covered with a 22by 40-mm cover slip, and counted with a low power objective (total magnification 30 to 40 diameters). When microfilaremia was low, 1 ml of blood drawn from the cephalic vein was concentrated by Knott's (1939) technique. Routinely, the MF in the least 3 preparations were counted (60 mm3 or 3 ml). For the exchange transfusions, blood from dogs without MF was cross-matched with blood from recipients and collected in acid-citrate-dextrose solution the day the experiments were done. The jugular vein and the femoral artery of the recipient