Judy W. Knight

Studies on a primaquine-tolerant strain of Plasmodium vivax from Brazil in Aotus and Saimiri monkeys.

A nonimmune American acquired an infection of Plasmodium vivax Type 1 malaria in Brazil in 1994. After returning to the U.S.A., he had a primary attack followed by 3 relapses. The primary attack and first 2 relapses were treated with a standard regimen of chloroquine, followed by 14 days of primaquine (15 mg/day). Following the third relapse, the primaquine treatment was extended to 28 days. No further relapses occurred. The lack of response to primaquine by this strain may recommend it as a suitable candidate for chemotherapeutic study if it can be adapted to an animal model. Anopheles quadrimaculatus mosquitoes infected by feeding on the patient during the first relapse were used to establish the strain in Aotus and Saimiri monkeys. Monkeys supported well the development of long-lasting parasitemia. Anopheles freeborni, Anopheles stephensi, and Anopheles gambiae mosquitoes were readily infected by feeding on the monkeys and by membrane feeding on diluted blood. Monkey-to-monkey transmission was obtained via the bites of infected mosquitoes and the intravenous injection of sporozoites dissected from salivary glands. This parasite is designated as the Brazil I/CDC strain of P. vivax.

Circulating antibody detection in human serum to mosquito salivary gland proteins by the avidin-biotin-peroxidase technique

Serum was collected from individuals with little, average, or extensive exposure to mosquito bites for determination of the presence of circulating antibodies to mosquito salivary gland proteins. Intensity of exposure was determined by mosquito-bite and exposure history. Sections cut through the thorax of four different species of mosquitoes, locally prevalent in South Florida, were exposed to serum and developed for antibody binding with the use of the avidin-biotin-peroxidase immunohistochemical technique. In subjects with histories of little or average exposure to mosquitoes, binding was observed that appeared to be species-specific; in subjects with extensive exposures, little antibody binding to salivary gland was noted. We hypothesize that suppression of the humoral immune response is an adaptive mechanism in response to significant exposures of mosquito salivary gland antigens.

Ultrastructural comparison of extracellular and intracellular encapsulation of Brugia malayi in Anopheles quadrimaculatus

Ultrastructural aspects of extracellular humoral encapsulation of microfilariae of Brugia malayi in the hemocoel of Anopheles quadrimaculatus were compared with those of intracellular encapsulation of first-stage larvae (L1) of the same parasite species, in the thoracic muscle cells of the same species of mosquito. The results showed that extracellular humoral encapsulation of microfilarial sheaths, and sheathed and exsheathed microfilariae, in the hemocoel of mosquitoes occurs around the parasite within the first 6 hr postingestion, apparently without initial participation of hemocytes. Hemocytes and their remnants were observed near the parasite during the first 6 hr postingestion. Within the next 24 hr, hemocytes attach to the initial humoral capsule. By contrast, intracellular encapsulation of L1S is initiated by the accumulation of a dense cytoplasmic layer derived from the infected thoracic muscle cell. Melanin deposits accumulate in this layer adjacent to the parasite cuticle, again without visible participation of hemocytes.

Plasmodium forresteri n. sp., from raptors in Florida and southern Georgia : Its distinction from Plasmodium elongatum morphologically within and among host species and by vector susceptibility

Plasmodium forresteri n. sp. naturally infects eastern screech-owls (Otus asio), great horned owls (Bubo virginianus), barred owls (Strix varia), bald eagles (Haliaeetus leucocephalus), red-shouldered hawks (Buteo lineatus), broad-winged hawks (Buteo platypterus), and red-tailed hawks (Buteo jamaicensis) in Florida and southern Georgia. Schizonts occur in mature or nearly mature erythrocytes, produce 2-6 merozoites arranged most commonly in fan or cruciform configuration, with mean dimensions among host species varying from 3.7 to 4.8 x 2.5 to 3.4 microns. Gametocytes are elongate, with mean dimensions among host species varying from 11.5 to 13.1 x 2.0 to 2.4 microns. One or both gametocyte margins are irregular and often crenulate. Gametocytes seldom fill the space between the erythrocyte nucleus and margin. Species characteristics were maintained in isodiagnostic Japanese quail (Coturnix japonica) and Pekin ducks (Anas platyrhynchos). In mosquito infection studies, only Culex restuans could support sporogony of P. forresteri, in contrast to Plasmodium elongatum of raptor origin that completed sporogony in both Cx. restuans and Culex nigripalpus.

Further characterization of refractoriness in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy)

Factors which control the expression of the refractory or susceptible condition to infection with Dirofilaria immitis in the mosquito. Aedes aegypti, were investigated using three protocols. (1) Microfilariae and prelarvae were injected into the hemocoel of susceptible A. aegypti. Some microfilariae and prelarvae developed to the L1 larval stage but they failed to complete development to the infective stage. (2) Enema of microfilariae and prelarvae from infected susceptible and refractory donor females were given into the midgut of uninfected susceptible and refractory recipient females. The results indicate that the conditions which inhibit the initiation of development are present in the Malpighian tubules and not in the midgut of the refractory mosquitoes. (3) Transplants of infected Malpighian tubules from susceptible and refractory donor females were made into the abdominal hemocoel of uninfected susceptible and refractory recipient females. The results showed that the refractory condition depends on the genetic makeup of the donor, not the recipient, mosquito. The above results taken as a whole indicate that the factors which control refractoriness are not present in the midgut but are present in the Malpighian tubule cells of refractory A. aegypti.

Vector ability of mosquitoes for isolates of Plasmodium elongatum from raptors in Florida.

Three isolates of Plasmodium elongatum were obtained from 3 species of raptors (red-tailed hawk [Buteo jamaicensis], bald eagle [Haliaeetus leucocephalus], and eastern screech owl [Otus asio]) from Florida using isodiagnostic techniques in Pekin ducks (Anas platyrhynchos). Six to 10 species of mosquitoes were tested for susceptibility to these 3 isolates. Complete development of the sporogonic cycle of the 3 isolates of P. elongatum occurred in 3 species of mosquitoes, Culex nigripalpus, Culex restuans, and Culex salinarius. The pattern of susceptibility was similar among the 3 isolates of P. elongatum in Cx. nigripalpus. Culex restuans and Cx. salinarius were significantly more susceptible than Cx. nigripalpus to the 3 isolates of P. elongatum tested. Culex nigripalpus transmitted all 3 isolates of P. elongatum from duck to duck both by bite and after intraperitoneal injection of sporozoites. Infections of the 2 isolates tested occurred in ducks after intraperitoneal injection of sporozoites from Cx. restuans and Cx. salinarius. The results suggest that these 3 Culex species are potential vectors of P. elongatum from raptors in Florida.

Temporal and geographic genetic variation in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Florida.

Abstract Culex (Culex) pipiens quinquefasciatus Say field population from Vero Beach, FL, sampled monthly over a period of 8 mo, a colony sample, and six geographic samples were analyzed for genetic variation at 12 enzymes (10 “neutral” gene enzymes with 11 putative loci and two “complex” gene enzymes) by using polyacrylamide gel electrophoresis. The analysis of the 11 putative loci in both temporal and geographic samples showed that the four loci (Gpi, Hk, Mdhp-2, and Pgm) diagnostic of Cx. p. quinquefasciatus in the southern United States are present in similar frequencies in Florida samples. The Cx. p. quinquefasciatus colony sample showed significantly lower genetic variation than the temporal field samples, measured by mean number of alleles per locus (colony 1.2 ± 0.1 versus field 1.44 ± 0.03), percentage of polymorphic loci (colony 18.2% versus field 28.4%), mean observed heterozygosity (Ho = colony 0.027 ± 0.02 versus field 0.09 ± 0.01), and mean Hardy-Weinberg expected heterozygosity (He = colony 0.025 ± 0.02 versus field 0.085 ± 0.01). Three of the 11 loci (Acoh, Pgd, and Pgm) from the Vero Beach field samples showed bimodal patterns in their frequencies of the most common allele during peak density of the population. The low value of Fst of 0.058 indicated minimum population substructuring among the temporal samples. Genetic variability values between geographic samples from the Florida panhandle and south Florida were not significant. Gene flow estimates based on FST = 0.05, indicating low levels of gene flow among the geographic samples of Cx. p. quinquefasciatus. The average Nei’s and modified Rogers’ genetic distances among the six populations were 0.005 ± 0.001 and 0.077 ± 0.007, respectively. The cluster analysis did not suggest geographic clustering. The analysis of the “complex” gene enzymes in both temporal and geographic samples of Cx. p. quinquefasciatus from Florida showed the presence of two highly amplified esterases (Estβ1 and Estα2\Estβ2), indicating resistance to organophosphate insecticides and highly amplified Aldox enzyme (an enzyme that indicates resistance to at least one insecticide and a herbicide). Comparison of our results with previous studies on Cx. p. quinquefasciatus populations in the United States indicates that the genetic characteristics of the Florida populations of Cx. p. quinquefasciatus are very similar to populations from areas where ecological conditions are very different.

Temporal and Geographic Genetic Variation in Culex nigripalpus Theobald (Culicidae: Diptera), a Vector of St. Louis Encephalitis Virus, from Florida

Abstract A field population of Culex (Culex) nigripalpus Theobald from Vero Beach, FL, sampled monthly over a period of 24 mo, a colony sample and 10 geographic samples were analyzed for genetic variation at 14 enzyme loci using polyacrylamide gel electrophoresis. The Cx. nigripalpus colony sample showed significantly lower genetic variation than the field-collected samples, measured by mean number of alleles per locus (colony 1.4 ± 0.1 versus field 2.1 ± 0.22), percentage of polymorphic loci (colony 35.7% versus field 54.8 ± 7.7%), but mean observed heterozygosity (Ho = colony 0.16 ± 0.07 versus field 0.17 ± 0.03) and mean Hardy–Weinberg expected heterozygosity (He = colony 0.14 ± 0.06 versus field 0.18 ± 0.02) did not differ significantly. Three of the 14 loci (Aldox, Gpd, and Gpi) from the Vero Beach field samples showed distinct temporal patterns in the frequency of the most common allele. Higher mean observed heterozygosity (Ho) occurred during months following high rainfall in the Vero Beach field samples than during months following low rainfall. The average Nm value of 3.6 indicated high gene flow among the temporally distributed samples of the Vero Beach population. Genetic variability values between geographic samples from Panhandle, FL and south Florida were not significant. Gene flow estimates based on FST = 0.039 provided a Nm of 6.2 indicating high levels of gene flow among the geographic samples of Cx. nigripalpus. The average Nei’s and modified Rogers’ genetic distances among the 10 populations were 0.009 ± 0.001 and 0.081 ± 0.004, respectively. The cluster analysis did not suggest geographic clustering. Because Cx. nigripalpus is the vector of St. Louis encephalitis (SLE) in Florida, temporal and geographic genetic variation in this species is discussed in relation to the seasonal and geographic SLE virus activity in Florida.

Selection of a strain of Aedes aegypti susceptible to Dirofilaria immitis and lacking intracellular concretions in the Malpighian tubules

Abstract In the process of selecting strains of Aedes aegypti refractory and susceptible to Dirofilaria immitis , we coselected for mosquitoes in the susceptible strain that lacked intracellular concretions in the cells of the Malpighian tubules. Using light and electron microscopy we were able to show that the cells lack both the crystals and the vacuoles surrounding them which are characteristic of the primary cells in the Malpighian tubules. Electron-probe microanalysis of whole tubules demonstrates that the tubules lacking crystals are also depleted in calcium, magnesium, manganese and phosphorus, compared to tubules from the refractory strain which contain crystals. Tests with larvae from these strains were unable to demonstrate increased mortality in larvae lacking intracellular crystals, either in media low in free calcium and magnesium, or in media high in zinc, manganese and magnesium. The genetic basis for the lack of crystals and the relationship of this trait to the susceptible condition are not yet known.

Ultrastructure of the intracellular melanization of Brugia malayi (Buckley) (Nematoda : Filarioidea) in the thoracic muscles of Anopheles quadrimaculatus (Say) (Diptera : Culicidae)

Ultrastructural details of the melanization of the filarial parasite Brugia malayi (Nematoda : Filarioidea) in the thoracic muscle cell of selected susceptible and refractory strains of Anopheles quadrimaculatus (Diptera : Culicidae) were examined. In both susceptible and refractory strains, following infection of the muscle cells, there is a dissolution of the sarcoplasmic cytoskeletal matrix surrounding the myofibrils, causing the myofibrils and mitochondria to drift apart and lose their regular spacing. This is coupled with a reorganization of the cytoplasm near the normally developing intracellular larva. Masses of vesicles and vesiculated cytoplasmic bodies accumulate near the larva. Eventually, these form a dense layer of cytoplasm around the parasite. Fine grains of melanin begin to appear in this dense cytoplasmic layer, increasing gradually until a nearly complete capsule of melanin surrounds the larva. The time course of melanization was found to be quite variable among and even within mosquitoes. Strains of A. quadrimaculatus selected to be refractory or susceptible to B. malayi, were found to vary substantially in the frequency of intracellular melanization, but not in the mechanism.