Role of habitat components on the dynamics of Aedes albopictus (Diptera: Culicidae) from New Orleans.

Monthly sampling of tire pile populations of Aedes albopictus (Skuse) in Orleans Parish, New Orleans, LA, was done in 1995 to determine prevalence of ascogregarine parasites and changes in wing length. Prevalence of Ascogregarina taiwanensis (Lien & Levine) infection was 100% in midsummer and decreased in the fall and spring (60-70%). Wing lengths were longest in the spring and fall and shortest in midsummer. We evaluated the effect of A. taiwanensis infections under high and deficient levels of leaf litter nutrients on mortality, development time, wing length, and reproductive potential of a New Orleans strain of Ae. albopictus. Parasitism and deficient nutrients caused a 35% increase in the rate of larval mortality and significantly extended the development time of females. Parasitized adults were 5% smaller and produced 23% fewer eggs than unparasitized siblings. In addition, abnormal Malpighian tubule morphology and melanization of ascogregarines were seen in adults from nutrient-deficient microcosms. We conclude that ascogregarine infections affect the dynamics of Ae. albopictus by increasing the mortality of immature stages when nutrients supplies are scarce, and by decreasing the reproductive capacity of females under high nutrient conditions.

Effect of nutrient levels and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) infections on the vector competence of Aedes albopictus (Diptera: Culicidae) for Dirofilaria immitis (Filarioidea: Onchocercidae).

The effect of habitat nutrients and Ascogregarina taiwanensis (Lein & Levine) infection on the vector competence of a New Orleans strain of Aedes albopictus (Skuse) for Dirofilaria immitis (Leidy) were evaluated. Larvae were infected with A. taiwanensis oocysts and reared under high and deficient nutrient conditions using leaf litter as a food source. Ascogregarine-infected and uninfected females were fed on D. immitis-infected blood and examined after 15 d for filariae. Susceptibility to infection with filaria was 60-70% for all females. In groups reared under high nutrients, the infective rate (3rd-stage larvae present) of coinfected females (18%) was significantly greater than females infected only with heartworm (6%). Host mortality following blood meals was significantly less in coinfected (22%) than in heartworm-infected females (37%). Under deficient nutrient conditions, there was no significant difference between the infective rate (8%) or post-blood meal mortality (5%) of coinfected females compared with heartworm-infected females. Prevalence of melanization reactions in coinfected females was significantly higher (31%) than in females infected only with heartworm (6%) at both nutrient levels. It is concluded that high nutrient levels and ascogregarine infection increase the vector competence of Ae. albopictus for D. immitis by enhancing the immune response so that fewer filariae develop, causing lower host mortality. Under low nutrient conditions, the smaller host contains less food reserves for filariae development, and in coinfected females melanization reactions and damage to the Malpighian tubules may reduce vector survival.

Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system.

The histopathologic and immunohistochemical features of early and late neuroborreliosis of the peripheral nervous system were investigated in rhesus macaques infected with the JD1 strain of Borrelia burgdorferi. Infection was proven by culture or polymerase chain reaction analysis of skin biopsies and indirectly by Western blot analysis. Three months after infection, neuritis involving multiple nerves was the most consistent neurologic manifestation. Both macrophages and B lymphocytes but not T lymphocytes were present in the cellular infiltrates. Axonal structures surrounding infiltrates had changes consisting of demyelination and axonal phagocytosis. Some of the Schwann cells in lesions stained with anti-nitrotyrosine and anti-tumor necrosis factor-alpha antibodies. B. burgdorferi, or antigens thereof, were visualized immunohistochemically within macrophages. Forty-six months after infection, the most common changes were regenerative, whereas neuritis was infrequent. Aberrant axonal regeneration, irregularly sized myelinated fibers, and fibrosis were frequently observed. Possible mechanisms to explain the appearance and subsidence of Lyme neuritis are discussed.

Histopathological, lymphoscintigraphical, and immunological changes in the inguinal lymph nodes of rhesus monkeys during the early course of infection with Brugia malayi.

The relationship of the early lymphatic pathophysiological alterations with those of tissue inflammatory and cellular responses in the inguinal lymph nodes of Brugia malayi-infected rhesus monkeys was examined. Each of five animals was inoculated subcutaneously in the right calf with 200 third stage larvae (L3) and 5 weeks later, before the onset of patency [10 to 12 weeks postinoculation (PI)], their right inguinal nodes began to show signs of enlargement, becoming most prominent between weeks 10 to 16 PI. Histopathologically, the right nodes had eosinophilic lymphadenitis, lymphoid hyperplasia, and pronounced germinal centers. Lymphoscintigraphy using 99mTc-antimony trisulfide colloid showed pathophysiological alterations of the lymph flow rate in the right leg but not in the left leg at weeks 7 and 15 PI. In vitro blastogenesis to B. malayi antigens at week 10 PI showed the inguinal lymph node cells proliferated more vigorously than did peripheral blood cells early in infection. However, at week 24 PI both lymph node and peripheral blood cells proliferated to antigens. Flow cytometry showed an upregulation of HLA-DR+ lymphocytes in right lymph node cells from infected animals when compared to those from control animals. No changes in CD2, CD4, CD8, CD20, CD29, and CD45R cell numbers in lymph node of infected animals were seen when compared to control animals. Our results show that lymphatic pathology occurs early before the onset of patency, correlating with a marked tissue inflammatory and cellular responses of lymph node cells in B. malayi-infected rhesus monkeys. The rhesus could be an extremely useful model for understanding the evolution of pathology and pathogenesis of the disease.

The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey.

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa.

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa-inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10-35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression of CD4+ and CD8+ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4+ and CD8+ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4+ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8+ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals (P < 0.0001), coinciding with an increase in CD8+ T cells numbers in these cultures. The data show that factors besides IL-2; and probably an imbalance in the percentages of CD4+ and CD8+ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa-inoculated rhesus monkeys.

Parasite antigenemia without microfilaremia in bancroftian filariasis.

The term "endemic normal" in the context of filariasis refers to people who are amicrofilaremic and free of clinical signs or symptoms of filariasis despite regular exposure to the parasite. Some sera from endemic normals contain soluble Wuchereria bancrofti antigens that are detectable by enzyme-linked immunosorbent assay. We now report evidence that filarial antigenemia in these people is not an artifact and that it is indicative of active W. bancrofti infection. Filarial antigenemia was first detected within one month of the onset of microfilarial patency in experimentally infected primates. Human sera from antigen-positive endemic normals contained the same filarial antigens (by Western blot) as sera from people with microfilaremia. Sera from antigen-positive endemic normals also contained significantly higher levels of immunoglobulin G4 antibodies to native and recombinant filarial antigens than sera from antigen-negative controls matched for age and sex. The epidemiology of filarial antigenemia in endemic normals was studied with sera from a population-based study of filariasis in an Egyptian village with a microfilaria prevalence of 29%. Seventeen percent of endemic normals had antigenemia, and this group comprised 11% of the total village sample. Filarial antigenemia was significantly more common in endemic normals more than 30 years of age than in younger people. These results suggest that amicrofilaremic and asymptomatic W. bancrofti infections are relatively common in endemic areas. Additional studies are needed to determine the clinical significance, prognosis, and optimal management of such infections.

Chronic lyme disease in the rhesus monkey.

BACKGROUND: We have previously reported the clinical, pathologic, and immunologic features of "early" Borrelia burgdorferi infection in rhesus monkeys (3). We have now evaluated these features during the chronic phase of Lyme disease in this animal model. EXPERIMENTAL DESIGN: Clinical signs, and pathologic changes at the gross and microscopic levels, were investigated 6 months post-infection in several organ systems of five rhesus macaques (Macaca mulatta), which were infected with Borrelia burgdorferi by allowing infected Ixodes scapularis nymphal ticks to feed on them. A sixth animal was used as an uninfected control. Borrelia antigens recognized by serum antibody were identified longitudinally by Western blot analysis, and C1q-binding immune complexes were quantified. Localization of the spirochete in the tissues was achieved by immunohistochemistry and in vitro culture. The species of spirocheta cultured was confirmed by the polymerase chain reaction. RESULTS: Chronic arthritis was observed in five out of five animals. The knee and elbow joints were the most consistently affected. Articular cartilage necrosis and/or degenerative arthropathy were the most severe joint structural changes. Synovial cell hyperplasia and a mononuclear/lymphocyte infiltrate were commonly seen. Nerve lesions were also observed, including nerve sheath fibrosis and focal demyelinization of the spinal cord. Peripheral neuropathy was observed in five out of five animals and could be correlated in the most severely affected monkey with the presence of higher levels of circulating immune complexes. Differences in disease severity did not correlate with differences in the antigens recognized on Western blot analysis. CONCLUSIONS: B. burgdorferi infection in rhesus macaques mirrors several aspects of both the early and chronic phases of the disease in humans. This animal model will facilitate the study of the pathogenesis of Lyme arthritis and neuroborreliosis.

Brugia malayi: the diagnostic potential of recombinant excretory/secretory antigens.

The diagnostic potential of recombinant E/S antigens of the lymphatic filaria Brugia malayi was investigated by Western blot. A cDNA expression library was constructed using B. malayi male adult worm mRNA, and E/S recombinants were identified with a rabbit antiserum raised against E/S products collected in vitro from B. malayi male and female adult worms. Two of these recombinants, Bm12 and Bm14L, were studied after subcloning the cDNA inserts in an Escherichia coli plasmid expression and purification vector, obtaining the inserts' nucleotide sequence, and purifying the expressed proteins. By homology of their deduced amino acid sequence with that of previously identified proteins, Bm12 was identified as the B. malayi gp 15/400 antigen, and Bm14 as a member of the hsp90 family of heat shock proteins. The antigenic cross-reactivity of the purified recombinant proteins was assessed with 28 serum samples from patients infected with Ascaris, Trichuris, or hookworm, and also with a few samples from patients with onchocerciasis and loiasis. For Bm12, the specificity for all of the intestinal helminthiasis together was 75%. Bm14L, on the other hand, cross-reacted with all of the ascariasis serum samples with which it was tested. Presence of antibodies cross-reactive with B. malayi was confirmed in all of these serum samples by examining their antibody reactivity with Western blots of extracts of whole B. malayi adult worms. A semiquantitative (+ or -) assessment of the sensitivity of Bm12 for antibody detection was performed using 6 serum samples from patients with chronic filariasis and 24 samples from patients with microfilaremia. All of these serum samples contained anti-Bm12 antibody (sensitivity of 100%). Finally, the ability of Bm12 to detect antibody before the onset of patency was established with a longitudinal collection of serum samples obtained from 2 African green vervets (Cercopithecus aethiops) and 3 rhesus macaques (Macaca mulatta), all of which were infected with B. malayi. Anti-Bm12 antibodies were detectable in all animals between 4 and 11 weeks before patency.

Effect of pyrimethamine resistance on sporogony in a Plasmodium berghei/Anopheles stephensi model.

A pyrimethamine-resistant line of Plasmodium berghei was derived by treating infected mice with high doses of pyrimethamine and selecting for recrudescence. This resistant line was compared with the parental pyrimethamine-sensitive line in order to ascertain whether drug resistance is associated with a biological advantage. Overall, the pyrimethamine-resistant line is quite similar to the sensitive line, except that it proceeds through sporogonic development more slowly than the pyrimethamine-sensitive parental line. However, under pyrimethamine pressure the sensitive line is unable to undergo the sporogonic cycle, whereas the resistant line is unaffected. These results indicate that the transmission of pyrimethamine resistance in this model is favored only under conditions of drug pressure.

Microfilarial densities, hematologic changes, and serum antibody levels in Loa loa-infected rhesus monkeys (Macaca mulatta).

To better define Loa loa infection in the rhesus monkey and assess the potential usefulness of this host as a model for studies of human loiasis, 12 monkeys (four splenectomized and eight nonsplenectomized) were inoculated with L. loa infective larvae. Microfilaremia and hematologic changes as well as parasite-specific antibody were assessed as a function of time in these animals. Eleven of 12 inoculated monkeys became microfilaremic. Splenectomized animals had moderate (250-1,000) to low (< 250) numbers of microfilariae per milliliter (mf/ml), whereas the mf/ml in nonsplenectomized animals varied from high (> 1,000) to low. A significant increase in total leukocyte and lymphocyte numbers was seen in animals with moderate-to-low mf/ml but not in animals with high mf/ml mainly because of variations between animals in the latter group, rather than a direct consequence of increased mf numbers. All infected animals developed an eosinophilia before patency, suggesting the adult worms most likely contribute to this phenomenon. As the infection progressed, the eosinophil numbers decreased significantly. Although splenectomized animals overall had slightly higher numbers of total leukocytes (lymphocytes and eosinophils), these hematologic changes as a function of time were not significantly different from those in nonsplenectomized animals. Parasite-specific IgG antibody was increased significantly before patency in all animals and with the exception of the one amicrofilaremic animal, it decreased after patency. This study shows that splenectomy of rhesus monkeys prior to L. loa inoculation does not enhance the microfilarial density nor does it adversely affect eosinophilia or antibody production.(ABSTRACT TRUNCATED AT 250 WORDS)

Early and early disseminated phases of Lyme disease in the rhesus monkey: a model for infection in humans.

We demonstrate that Borrelia burgdorferi infection in the rhesus monkey mimics the early and early disseminated phases of human Lyme disease. Clinical, bacteriological, immunological, and pathological signs of infection were investigated during 13 weeks after inoculation of the spirochete. Three animals were given B. burgdorferi (strain JD1) by needle inoculations, six animals were exposed to the bite of B. burgdorferi-infected Ixodes dammini ticks, and three animals were uninfected controls. B. burgdorferi could be recovered from all animals that were given the spirochete. Bacteria were detectable until week 6 postinoculation (p.i.) in blood, until week 8 p.i. in skin biopsies, and at 10 weeks p.i. in the conjunctiva of one of two animals which developed conjunctivitis. Erythema migrans (EM) appeared in one of the three animals infected by needle inoculation and in five of the six animals infected by ticks. Deep dermal perivascular lymphocytic infiltrations (characteristic of human EM) were observed in all animals showing EM clinically. Both EM and conjunctivitis were documented concomitantly with the presence of the spirochete. Lethargy, splenomegaly, and cerebrospinal fluid pleocytosis were also noted in some animals, but the direct connection of these signs with the infection was not shown. The appearance rate of immunoglobulin M and immunoglobulin G antibodies to B. burgdorferi, as well as the antigen spectra recognized, were remarkably similar to those seen in humans. Serum antibodies from infected animals were able to kill B. burgdorferi in vitro in the presence of rhesus complement. The rhesus monkey model appears to be useful for the investigation of the immunology and pathogenesis of Lyme disease and for the development of immunoprophylactic, diagnostic, and chemotherapeutic protocols.

Gametogenesis and sporogony of Hepatozoon mocassini (Apicomplexa: Adeleina: Hepatozoidae) in an experimental mosquito host, Aedes aegypti.

The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti, an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiation were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a parasitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporozoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endoplasmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.

Mansonella ozzardi: the course of patency in experimentally-infected patas monkeys.

Twenty-five of 30 patas monkeys (Erythrocebus patas) inoculated with varying numbers (35 to 135) of third-stage larvae of Mansonella ozzardi developed patent infections in an average of 163 days. There was no correlation between the size of the inoculum and the length of the prepatent period. Ten of the monkeys were monitored thereafter by regular blood examination for extended periods of time in order to characterize the onset, course and duration of patency. Typically, with the onset of patency, microfilaremias increased steadily, peaking at about 20 weeks and then decreased slowly stabilizing at low levels for up to 48 weeks. Thereafter microfilariae disappeared from the blood and occasionally reappeared in scanty numbers. Laparotomies and followup studies indicated that the spleen was involved in the suppression of peripheral microfilaremia as had been observed earlier in patas monkeys infected with Loa loa. In ten monkeys splenectomized after the initial "wave" of microfilaremia, it was observed that (a) 30% of the animals remained amicrofilaremic, (b) another 30% reestablished patent infections but microfilaremias were lower than presplenectomy levels, and (c) in the remaining 40%, levels of microfilaremia equaled or exceeded pre-splenectomy levels. Patent infections persisted for up to 212 weeks. One monkey splenectomized prior to inoculation with 87 larvae developed a patent infection with microfilaremia which persisted for 156 weeks. Three monkeys with low and high levels of microfilaremia bled at four hour intervals over a 28 hour period showed no evidence of periodicity in the microfilaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypnozoites of Plasmodium simiovale.

Hypnozoites of Plasmodium simiovale were detected in liver biopsies from a rhesus monkey (Macaca mulatta) inoculated eight days previously with sporozoites from heavily infected anopheline mosquitoes. The tissue forms, 6 mu in diameter, were found within the cytoplasm of hepatic parenchymal cells by immunofluorescence and restained with Giemsa. This is the first report of latent, pre-erythrocytic stages from an ovale-type relapsing malaria.

Poor vector efficiency of Culex quinquefasciatus following infection with Dirofilaria immitis.

The vector efficiency of the Haiti and Covington strains of Culex quinquefasciatus after feeding on dog blood infected with Dirofilaria immitis (approximately 110 microfilariae (mf)/20 microliters of blood) was 1.2 and 0.3%, respectively, versus nearly 20% for the Vero Beach strain of Aedes taeniorhynchus. At a much higher microfilaremia (approximately 400 mf/20 microliters), it was 1.6, 0.5 and 31.5%, respectively. The poor vector efficiency of the 2 Cx. quinquefasciatus strains probably was due to the formation of long, needle-like oxyhemoglobin crystals in the blood meal, which prevented the migration of microfilariae to the Malpighian tubules. Crystals did not form when Cx. quinquefasciatus ingested D. immitis microfilariae mixed in human blood.

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears.

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema viteae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanol and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection of filarial infections, is discussed.

Differential humoral and cellular immunoreactivity to saline- and detergent-extracted filarial antigens.

The immunoreactivity to saline- and detergent-extracted filarial antigens of 31 Haitian subjects exposed to or infected with Wuchereria bancrofti was analysed. Cellular reactivity, monitored by blastogenesis in vitro, was significantly greater to a soluble extract of Brugia pahangi than to a detergent-extracted antigenic preparation. In contrast, serum antibody levels against the detergent extract were significantly higher. These differences were consistently observed in symptomatic, microfilaraemic, and asymptomatic/amicrofilaraemic groups of patients. These patterns of immunoreactivity may reflect intrinsic differences between the 2 antigenic preparations in terms of composition or their mode of presentation to the host immune system.

Uptake and development of Wuchereria bancrofti in Culex quinquefasciatus that fed on Haitian carriers with different microfilaria densities.

In studies conducted between 1984 and 1986, the vector competency of Culex quinquefasciatus was assessed after bloodfeeding on 61 Haitian volunteers with different densities of Wuchereria bancrofti microfilariae (mf) and on 11 that were amicrofilaremic. Infected volunteers included persons previously given diethylcarbamazine citrate for 12 consecutive days and some that were untreated. Mosquitoes, derived from field-collected larvae, were released under bed nets and fed upon the volunteers while they slept. The mean mf uptake in mosquitoes that fed on 21 carriers with low to ultralow densities (1-28 mf/ml blood) was 0.2-4 mf/mosquito (mean = 1.7 mf). The observed infectivity rate of greater than 3,000 mosquitoes that fed on high (56-7,500 mf/ml blood; median = 525), low (11-49 mf/ml blood; median = 20), or ultralow (1-10 mf/ml blood; median = 3) density carriers was 4, 11, and 30 times higher, respectively, than the expected rate calculated from the estimated volume of the imbibed bloodmeal. These results indicate that "hidden" carriers (less than 50 mf/ml blood) may serve as a source of infection for mosquitoes, and support the increasing evidence that mosquitoes ingest more microfilariae than expected.