Genomics of Loa loa, a Wolbachia-free filarial parasite of humans.

Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, L. loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4-Mb genome of L. loa and that of the related filarial parasite Wuchereria bancrofti and predict 14,907 L. loa genes on the basis of microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to those of several other nematodes, we demonstrate synteny among filariae but not with nonparasitic nematodes. The L. loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for use in humans. Despite lacking Wolbachia, L. loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role of Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and nonparasitic nematodes.

Filarial parasites develop faster and reproduce earlier in response to host immune effectors that determine filarial life expectancy.

Humans and other mammals mount vigorous immune assaults against helminth parasites, yet there are intriguing reports that the immune response can enhance rather than impair parasite development. It has been hypothesized that helminths, like many free-living organisms, should optimize their development and reproduction in response to cues predicting future life expectancy. However, immune-dependent development by helminth parasites has so far eluded such evolutionary explanation. By manipulating various arms of the immune response of experimental hosts, we show that filarial nematodes, the parasites responsible for debilitating diseases in humans like river blindness and elephantiasis, accelerate their development in response to the IL-5 driven eosinophilia they encounter when infecting a host. Consequently they produce microfilariae, their transmission stages, earlier and in greater numbers. Eosinophilia is a primary host determinant of filarial life expectancy, operating both at larval and at late adult stages in anatomically and temporally separate locations, and is implicated in vaccine-mediated protection. Filarial nematodes are therefore able to adjust their reproductive schedules in response to an environmental predictor of their probability of survival, as proposed by evolutionary theory, thereby mitigating the effects of the immune attack to which helminths are most susceptible. Enhancing protective immunity against filarial nematodes, for example through vaccination, may be less effective at reducing transmission than would be expected and may, at worst, lead to increased transmission and, hence, pathology.

CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to filarial parasites in vivo.

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.

Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution.

Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.

Parasitic helminth genomics. Filarial Genome Project.

The initiation of genome projects on helminths of medical importance promises to yield new drug targets and vaccine candidates in unprecedented numbers. In order to exploit this emerging data it is essential that the user community is aware of the scope and quality of data available, and that the genome projects provide analyses of the raw data to highlight potential genes of interest. Core bioinformatics support for the parasite genome projects has promoted these approaches. In the Brugia genome project, a combination of expressed sequence tag sequencing from multiple cDNA libraries representing the complete filarial nematode lifecycle, and comparative analysis of the sequence dataset, particularly using the complete genome sequence of the model nematode C. elegans, has proved very effective in gene discovery.

Defense reactions of mosquitoes to filarial worms: effect of host age on the immune response to Dirofilaria immitis microfilariae.

The melanization response of Aedes aegypti black-eyed Liverpool strain (LVP) and Aedes trivittatus against intrathoracically inoculated Dirofilaria immitis microfilariae (mff) was assessed in mosquitoes less than 1, 14, 21, and 28 days after adult ecdysis. There was a significant decrease in the melanization response of A. aegypti 14 days of age and older at 1, 3, and 5 days postinoculation (PI) compared to less than 1-day-old mosquitoes. The response also was reduced significantly in 14- to 28-day-old A. trivittatus on days 1 and 3 PI. Although essentially 100% of recovered mff were melanized by day 5 PI in A. trivittatus, the amount of melanin deposited was much less than that seen in 0-day-old mosquitoes. Potential mechanisms responsible for a reduced immune competence in older mosquitoes and the possible relationship to vector potential are discussed.

Cryopreservation of third-stage larvae of Brugia malayi and Dipetalonema viteae.

Methods are described for the cryopreservation of third-stage larvae of Brugia malayi. Optimum conditions utilized larvae free from the mosquito host frozen at the rate of -1 degree or -0.8 degrees C per min in medium containing 9% dimethyl sulfoxide and 0.004 M polyvinylpyrrolidone. Nonfrozen or thawed larvae were inoculated intraperitoneally into jirds (Meriones unguiculatus), the thawed larvae after cryogenic storage for 5-378 days. In general, the percentage of adult worms recovered at necropsy was comparable between the two groups and ranged from a mean of 6-9% of the larval inoculum. In addition, three of four patas monkeys (Erythrocebus patas) inoculated with thawed B. malayi larvae developed patent infections. The cryopreservation of third-stage larvae of Dipetalonema viteae also is discussed.

The experimental transmission of Parafilaria bovicola to cattle in South Africa using Musca species (subgenus Eumusca) as intermediate hosts.

In controlled experiments in an insect-free stable, cattle became infected with Parafilaria bovicola when Musca lusoria, infected with the larvae of this worm, were allowed to feed on a fresh skin incision, and when infective larvae were placed on fresh skin incisions, injected subcutaneously or into the jugular vein, or instilled into the eyes. The sites of blood spots caused by ovipositing P. bovicola females and the sites of carcass lesions were seldom close to the site of infection, an indication that the worms had migrated. The prepatent period of P. bovicola in 4 cattle which developed blood spots ranged from 242--319 days. Neither of the infected cattle that were kept continuously in a shady stable showed blood spots, but 4 out of 7 infected cattle which spent some time in the sun bled. However, carcass lesions on shaded cattle were similar in appearance to those on cattle kept outdoors. Infective larvae were stimulated to escape from the mouth-parts of infected M. lusoria and Musca xanthomelas s.s. when these were fed citrated ox blood warmed to 38--40 degrees C. No escape took place when the flies were fed warmed saline or warmed 15% sucrose solution.

[Diaplacental transmission of microfilaria of the species, Brugia pahangi, in the cat]. / Diaplacentare Ubertragung von Mikrofilarien der Art Brugia pahangi bei der Katze.

Eight kittens born of two Brugia pahangi infected cats have been studied for transplacental passed microfilariae. In the peripheral blood microfilariae could not be demonstrated at any time. However, in the lung of a young cat killed two days post partum ca. 30 microfilariae have been found (microfilaremia of the mother 90 mf/20 mm3). Histological studies suggested two possibilities of transplacental passage--by blood and by secretion of the uterus glands.

Transovarially-transmitted intracellular microorganisms in adult and larval stages of Brugia malayi.

Observation of intracellular organisms in the lateral chords of Brugia malayi adults initiated further studies to determine the prevalence of these organisms within the tissues of adult worms and of larvae. The organisms were found in the lateral chords of adult males and females, microfilariae, first-, second-, third-, and fourth-stage larvae. In the females, they were present in the oogonia, oocytes, and developing eggs, suggesting transovarial transmission within the life cycle of the filarid. The organisms may have a developmental cycle consisting of more than one stage, including a small spheroidal stage up to 0.6 micrometer in size and a larger form up to 1.5 micrometer in length, all of which occur in the cytoplasm within a vesicle formed of host membrane. Each stage lacks a definite cell wall, being bound by 2 trilaminate membranes. The bacterial entities in B. malayi resemble both in morphology and development the organisms found in other filarids, but whether they affect the vertebrate host in any way remains to be determined. Their presence within certain cells of the developing eggs could be exploited as intracellular markers for the organogenesis of the lateral chords and the ovary.

Experimental infection of Rattus sabanus and Rattus muelleri with subperiodic Brugia malayi.

R. sabanus and R. muelleri are very common in the lowland forests of Malaysia. In nature they are infected with Breinlia sp. and D. ramachandrani. In an attempt to determine whether they are also susceptible to subperiodic B. malayi and thereby being potential reservoirs of infection of the disease, 24 R. muelleri and 17 R. sabanus were experimentally infected with the parasite. Results show that although they can support the full development of the parasite, they are poor hosts. This confirms the observation that in Malaysia natural infection of Rattus spp. with the parasite has not been seen. These rats therefore are probably not important in the zoonotic transmission of subperiodic B. malayi in Malaysia.